What’s Known

The next-generation technique is well known for whole-genome sequencing to identify bacterial genomic characteristics, including antibiotic resistance genes. Resistance genes were mentioned in previous studies but not identified in Iraq.

What’s New

This is considered the first study to know the bacterial whole genetic sequence in Iraq, rather than being the first study to draw the complete genetic map of this bacterium. Moreover, it is the only study in Iraq to identify prophage and CRISPER-cas system, insertion sites, and bacterial serotype in the genetic sequence, and finally, the first to draw the genetic tree comparing our isolate to other countries.

Introduction

About 30% of Gram-negative infections are caused by Klebsiella pneumoniae, ¹ a non-motile Gram-negative encapsulated opportunistic bacterium. ² which encompasses plentiful chromosomal and plasmid-encoded antibiotic resistance genes (ARGs K. pneumoniae is one of the leading causes of community-acquired infections, including urinary tract, respiratory, and bloodstream infections. ³ Its distribution is a major public health problem, especially with the emergence of multidrug-resistant (MDR) strains. ⁴ Another research has demonstrated antibiotic resistance in K. pneumoniae, which poses a significant and immediate threat. ⁴ Such bacteria have developed resistance to antimicrobial agents by several mechanisms, including alterations in permeability, active efflux, enzymatic modification, degradation, modification of antibiotic targets, acquisition of alternate metabolic pathways, and overproduction of the target enzyme. ⁵

This pathogenic microorganism induces infection by various virulence factors, including surface antigens, fimbriae, iron uptake, capsule, serum resistance, outer membrane proteins, toxins, and siderophores, which enable the bacteria to penetrate and proliferate within the host. ⁶

Genotyping is essential for identifying this significant pathogen. Multilocus Sequence Typing (MLST) is a molecular technique to determine the genetic association between bacterial isolates. It is mainly used for molecular epidemiological studies of microorganisms that are of public health concern. ⁷ MLST analysis has demonstrated that K. pneumoniae exhibits a predominantly oligoclonal nature, with a plenty of sequence types (ST) identified, including ST 11, 14, 15, 26, 101, 147, 149, 231, 258, 627, and 977. ⁸ Several STs have exhibited geographic specificity, with some becoming both endemic and/or epidemic. ⁹

The K. pneumoniae capsule is an intricate formation consisting of 16 to 20 various genes, depending on the serotype. Moreover, the capsule functions as a protective barrier for the host’s initial inflammatory response by inhibiting opsonization. ¹⁰

K. pneumoniae can produce two main porins, OmpK35 and OmpK36. Porins are proteins that enable the movement of hydrophilic substances in both directions across the cell membrane. Due to the lack of outer membrane porins, K. pneumoniae exhibits enhanced resistance to cephalosporin and carbapenem, and reduced pathogenicity. ¹¹

Whole-genome sequencing (WGS) offers unprecedented resolution and comprehensive information for the entire bacterial genetic material, encompassing all key genomic elements. Beyond bacterial identification and molecular characterization, WGS holds significant potential for predicting microbial phenotypes. ¹²

The objective of the study was to analyze the key phenotypic and genetic features present in the clinical K. pneumoniae isolate. It is the first study in Iraq to utilize WGS for a comprehensive characterization of an opportunistic pathogen, thus emphasizing the need to use whole-genomic sequencing to track the development of resistance and virulence patterns in clinical strains of K. pneumoniae.

Materials and Methods

Bacterial Isolation and Identification

Ninety-one K. pneumoniae isolates were obtained from urine samples collected from Al-Imamian Al-Kadhimiyain Medical City (IKMC) between 2022 and 2023 from children under twelve years old. Primary isolation was performed using MacConkey agar as a selective culture medium. Subsequent identification and confirmation of the isolates were conducted using the VITEK-2 (bioMérieux, Inc., Durham, NC) automated microbial identification system. The strains were subcultured on Luria-Bertani (LB) or DNA extraction and incubated at 37 °C for 24 hours under aerobic conditions.

DNA Extraction

One strain isolated from a 4-day-old infant due to its extreme drug-resistant profile. It was centrifuged at 5000 x g for 10 min to obtain bacterial cultures. Following the manufacturer’s procedure for bacterial samples, the DNA was isolated using the QIAamp DNA Mini kit (QIAGEN, Germany). The separated DNA was kept at a temperature of -20 ˚C until it was time to prepare the library.

Whole-Genome Sequencing (WGS)

Genomic DNA was extracted, followed by WGS on the Illumina HiSeq 2000 platform (Illumina, USA). De novo genome assembly was performed using SPAdes (software developers: Russia and USA) (https://ablab.github.io/spades/). Draft genomes were annotated using rapid prokaryotic genome annotation (Prokka) (https://training.galaxyproject.org/training-material/). The resulting K. pneumoniae genome exhibited high data quality and was submitted to the National Center for Biotechnology Information (NCBI).

Data Accessibility

The sequence data have been successfully submitted to the NCBI’s database with the BioProject Accession Number PRJNA1130523 () and BioSample Accession Number SAMN42208297 (https://www.ncbi.nlm.nih.gov/biosample/42208297).

Genome Mapping

The CGView Server (https://js.cgview.ca/) was used to produce a comparative circular genome map, with the Klebsiella pneumoniae strain HS11286 genome sequence (Accession number: CP003200.1) acting as the reference.

Multilocus Sequence Typing

The following seven housekeeping genes were used to do multilocus sequence typing (MLST): gapA, infB, mdh, pgi, phoE, rpoB, and tonB. The Pasteur Institute’s BIGS database (https://bigsdb.pasteur.fr/) was used to identify allele sequences and STs, reporting only exact allelic profile matches.

Antibiotic Resistance

Bacterial resistance genes and plasmid replicons were identified using the ResFinder tool (http://genepi.food.dtu.dk/resfinder) with a 100% identity and an 80% minimum match length.

Virulence Factors

The previously obtained K. pneumoniae genome was analyzed using the Virulence Factors Database (http://www.mgc.ac.cn/VFs/) to identify known virulence genes. Clusters examined included fim (A-H, and K), mrk (A-D, and F), ecp (A-E), and genes for outer membrane proteins (ompA, ompK35, ompK36), the virB cluster (1-11) of the T4SS, and hypermucoviscosity genes magA and rmpA. Additionally, siderophores and enterobactin genes coded by fes, salmochelin coded by iroN and fyuA, and iut clusters, coding for yersiniabactin and aerobactin, respectively, were investigated. Gene queries were based on the CP003200 reference strain, using an identity threshold of 100% and a minimum match length of 80%.

16SrRNA Phylogeny

16S rRNA sequences from our isolate genome sequence were aligned with the NCBI database using Molecular Evolutionary Genetics Analysis (MEGA11) (Pennsylvania State University, USA) (https://www.megasoftware.net/). These sequences were used to construct neighbor-joining trees using the MUSCLE tool in MEGA11. The extracted tree was edited using the iTOL server (https://itol.embl.de/).

Identification of Mobile Genetic Elements, CRISPR-Cas Region, and Bacterial Serotype

The ISFinder server (https://isfinder.biotoul.fr/about.php) was used to detect Other Mobile Genetic Elements by comparing the FASTA to the database. The phage regions were identified and visualized using the PHASTER tool (https://phaster.ca/), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) were identified and visualized using the CRISPRFinder tool (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index). The Kaptive tool (https://kaptive-web.erc.monash.edu/jobs) was employed for capsular typing. It was utilized to identify the K/O capsular type of our isolate. Furthermore, the plasmids were characterized by aligning the contigs of the sample with the plasmid database, and the reads were aligned with the PlasmidFinder database (http://genepi.food.dtu.dk/resfinder).

Single Nucleotide Polymorphisms (SNP) and Indel Discovery

Variant Count: The generated WGS data were utilized to identify genetic variations using the NCBI reference genome. Following the elimination of duplicates using Sambamba and the identification of variants using SAMTools (https://sourceforge.net/p/samtools/mailman/samtools-devel/thread/2F0E69A8-A2DD-4D6E-9EDE-2A9C0506DA0F@sanger.ac.uk/), data for each variant was collected and categorized based on their respective chromosomes or scaffolds.

Results

The genomic sequences of the bacterial strain were obtained, which were confirmed to be K. pneumoniae with serotype K36:O2a and ST437. The genomes had an average assembly length of 2,192,701,200 base pairs, 14,521,200 reads, a GC content of 57.55%, and 7,604 Coding DNA Sequences (CDS).

16SrRNA was analyzed and compared with the NCBI database to conduct the molecular characterization and phylogenetic distance tree of K. pneumoniae, revealing that our strain is more genetically related to the USA and India than Iraq (figure 1).

Figure 1. A phylogenetic tree based on nucleotide sequences from the 16S rRNA gene of Klebsiella pneumoniae was constructed using MEGA11, with sequence alignment performed through MUSCLE. Bootstrap values, expressed as percentages, were calculated from 1000 resamplings and are displayed at the corresponding nodes in the cladogram.

The data analysis revealed the existence of 54 antimicrobial resistance genes that are accountable for the bacterium strain’s resistance to different categories of drugs, as shown in table 1. The antibiotic classes identified in our analysis as being resistant are β-lactamases, aminoglycosides, phenicols, macrolides, sulphonamides, quinolones, tetracycline, trimethoprim, Colistin, fosfomycin, and glycopeptides. Antibiotic efflux, target replacement, and antibiotic inactivation are three resistance mechanisms caused by the genes shown in Supplementary table 1.

The allele sequences and STs of our K. pneumoniae isolate revealed that the strain belongs to the ST437 type; this study is the first to detect this serotype in Iraq. Here, for the first time, we describe a capsular polysaccharide (CPS) cluster for a clinical isolate of K. pneumoniae from Iraq. The collected data, including K-serotyping, confirmed the presence of 22 unique CPS genes and 10 unique lipoprotein (LPO) genes (figure 2), which classified our isolate as belonging to the K36:O2a capsular serotype.

Figure 2. The gene locus of the isolate was analyzed in comparison to the reference genes in the Kaptive database. (a) The K-locus gene arrangement of the Klebsiella pneumoniae isolate is displayed, with the gene names indicated above the colored arrows. (b) The O-locus gene arrangement of the Klebsiella pneumoniae isolate is also shown, with gene names labeled above the colored arrows. Lighter-colored arrows represent genes with low identity. The colors were fixed by the server

The allele sequences and STs of our K. pneumoniae isolate revealed that the strain belongs to the ST437 type. This study is the first to detect this serotype in Iraq.

The analysis of virulence genes indicated the presence of the mrk cluster, as shown in supplementary table 2. It supports the idea that type 3 fimbriae are a characteristic feature of K. pneumoniae virulence. In addition, the strain harbored a total of fifteen plasmid replicons belonging to the incompatibility types shown in table 2.

The PHAge Search Tool Enhanced Release (PHASTER) was applied to determine whether the bacteriophages were present in the sample. The results of the prophage regions for our sample are shown in figure 3. Our sample included four intact bacteriophages and two questionable ones, which were reported for the first time in Iraq. The intact bacteriophages identified were Edward_GF, Klebsi_ST512_KPC3phi13, Salmon_SEN34, and Klebsi_ST147_VIM1phi7. The questionable prophages identified were Entero_P4 and Salmon_ST64B. One CRISPR array was detected in the sample using CRISPRFinder. To our knowledge, this result is the first to detect the CRISPR-Cas system in Iraq. The CRISPRs in the sample were non-Cas-associated, as shown in figure 4. About 10293 Single Nucleotide Polymorphisms (SNPs) were detected in our isolate, including 6936 transitions and 3357 transversions, 61 insertions, and 71 deletions, causing 3707 protein frameshifts and 909 silent mutations.

Figure 3. The circular genomic map of our isolate illustrates various features: the black histogram represents the GC content, while the blue-red histogram depicts the GC skew distortion. The reference genome is shown as a dark purple ring, and the coding sequences (CDS) in the isolate are represented by a green ring. The open reading frames (ORFs) are displayed as a light purple ring. Red arrows indicate resistance genes, whereas orange arrows denote horizontal gene transfer (HGT) events.

Figure 4.A CRISPR array was identified in the Klebsiella pneumoniae K36:O2a isolate, consisting of two direct repeat sequences and one spacer sequence, located between nucleotides 1,158,275 and 1,158,370.

Figure 5 shows a circular genomic map of our strain, conducted using a GC viewer. It shows GC content, GC skew, and CDS content in the K. pneumonia K36:O2a ST437 strain. Seven insertion sequences, revealed with the analysis using ISfinder, are shown in table 3 as part of other mobile genetic elements.

Figure 5. A circular genomic map of the Klebsiella pneumoniae isolate was generated using PHASTER, highlighting the regions of phages. The analysis identified a total of 4,428 prophage genes distributed across four intact prophages and two incomplete prophages.

Discussion

The results of this study showed the genetic similarity of our bacterial isolate to strains from the USA and India rather than Iraq. This might be due to various factors, such as global travel ¹³ and human migration. ¹⁴ Despite a relatively small number of patients, medical tourism has grown in popularity. There have been reports of the ST437 K. pneumoniae in North America, Europe, and Asia. ¹⁵ In the Indian subcontinent, New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae have been reported to have spread globally. ¹⁶ Furthermore, MDR bacteria are a significant clinical concern due to their ability to acquire more 16S rRNA methyltransferase genes, which confer exceptionally high resistance to various aminoglycosides. ¹⁷

The WGS of our isolate revealed a large number of resistance genes. This finding contradicts previous reports on other strains of K. pneumoniae, including ST152 and ST17, which carried 48 and 36 resistance genes, respectively. ¹⁸

The primary resistance mechanism detected is the deactivation of antibiotics through the aph genes, which encode aminoglycoside phosphotransferase, and the aac genes, encoding aminoglycoside acetyltransferase. The acetyltransferase, aad genes, and ant genes are responsible for aminoglycoside resistance. This agrees with a study conducted by Ramirez and Tolmasky in 2010. ¹⁹ Additionally, nucleotidyltransferase plays a significant role in aminoglycoside resistance. ²⁰ The substitution of the antibiotic target is mediated by sul1, which encodes a dihydropteroate synthase (DHPS), leading to the bacterial reduced affinity for sulfonamides. The sul genes were demonstrated at a high rate, especially the sul1 gene, when a study conducted in Iran revealed that 71% of K. pneumoniae isolates harbored sul1. ²¹ dfr genes are responsible for carrying the trimethoprim resistance dfrA12 and dfrA30 detected in our isolate and found in a previous European study. ²² The process of rRNA methylase is utilized in conferring resistance to macrolides through the erm genes, which are a widespread mechanism of resistance to the macrolides. ²³ Methionine reductase genes (msr) were detected in this study. Our findings aligned with the Department of Environmental & Occupational Health Sciences database published by M. C. Roberts (http://faculty.washington.edu/marilynr/); the msr enzymes are important factors in protecting bacterial species against oxidative stress and in contributing to their pathogenicity. ²⁴ To our knowledge, this was the first study to detect K. pneumonia’s msr genes in Iraq. Further studies are needed to detect this gene cluster and to understand the necessity of bacteria for those enzyme-coding genes, their regulation, and their precise function in physiology, antibiotic resistance, and bacterial virulence. The tet and oqxA,B genes present efflux pump ability. The tet gene is responsible for conferring tetracycline resistance; a recent investigation on surveillance revealed that around 67% and 45% of E. coli and Klebsiella species (spp.) in some European nations were resistant to tetracycline. ²⁵ The antibiotic resistance frequency was detected in our isolate’s genome with 100% identity for the oqxA and oqxB genes. These genes are responsible for conferring resistance to quinolones. Another researcher observed a significant resistance to this family of antibiotics. ²⁶ While bla genes are responsible for β-lactamase production our isolate harbors blaCTX-M-15, blaNDM-5, blaOXA-181, blaSHV-182, blaTEM-1B genes beside ampH gene, two enzymes involved in low-level beta-lactam resistance are often chromosomally incorporated in Klebsiella spp.; two genes, particularly TEM-1B, have been identified as the most commonly found β-lactamase genes in K. pneumoniae. ²⁷ fos genes confer fosfomycin resistance through a pathway involving glutathione transferase; it is expected that these genes were shown to be prevalent as it has been globally documented that fosfomycin is frequently used in combination with aminoglycosides for the treatment of numerous urinary tract infections. ²⁸ Furthermore, antibiotic resistance has been on the rise in Iraq due to the overuse and misuse of antibiotics. In 2021, Al-Taie and colleagues found that 45.8% of individuals were engaged in self-medication of antibiotics without a prescription. ²⁹ Clinical samples of K. pneumoniae may contribute to the continued ability of this sequence type to evade various classes of antibiotics, as shown by the diverse array of resistance genes and mechanisms identified in this study. ¹⁸

The capsular serotypes are known to be strongly related to hypervirulence in K. pneumoniae. ³⁰ Our results are in line with previous research showing that certain serotypes of K. pneumoniae have di-mannose/rhamnose residues, and that genetically modified strains that produce capsules are less able to stimulate polymorphonuclear leucocytes (PMNs). ³¹

The strain exhibited both the ompA and ompK35 genes. The higher occurrence of ompK35 than ompK36 indicates that ompK35 plays a crucial function in maintaining the structural integrity of the outer membrane as a porin. On the other hand, the absence of ompK36 enhances antibiotic resistance, decreases bacterial fitness, and diminishes virulence. ³² Specifically, the blaOXA carbapenemase gene was found in more than 70% of K. pneumoniae strains that lacked the ompK36 gene, pointing to a possible connection between ompK36 porin gene deficiency and carbapenem antibiotic resistance. ³³ K. pneumoniae’s production of siderophores during infection affects the localization of tissues, the infection’s spread throughout the body, and the host’s survival. ³⁴ The presence of the Enterobactin genes in the K. pneumoniae genome is expected, as catecholate is commonly encoded in the core genome of K. pneumoniae. ³

The presence of the IncFIB plasmid, a conjugative plasmid, has been associated with the transmission of several genes in K. pneumoniae, including blaNDM-1, blaSHV-12, blaCTXM-15, and blaOXA-1. ³⁵ Klebsiella pneumoniae is the dominant species carrying colistin‐resistance-inducing insertion sequence elements on plasmids. ³⁶ SKpn1 belongs to the IS3 family and is known for its transposition activity between plasmids and chromosomes of K. pneumoniae and E. coli. ³⁷ Since complete prophages are typically subject to quick deletion from bacteria, it was anticipated that incomplete and questionable prophages would be much more common; instead, our sample obtained four intact bacteriophages and two questionable ones observed for the first time in Iraq. The intact bacteriophages identified were Edward_GF, Klebsi_ST512_KPC3phi13, Salmon_SEN34, and Klebsi_ST147_VIM1phi7. The questionable prophages identified were Entero_P4 and Salmon_ST64B. This finding contrasts with the study conducted by Kang and colleague in 2023, which reported the lack of Klebsi_ST512_KPC3phi13 and Entero_P4 prophages. ³⁸ No virulence factors (VF) and antimicrobial resistance (AMR) genes were detected in the prophage sequences found in our isolate. In another study, a large number of VF and AMR genes were found in prophages, ³⁸ which showed that plasmids may not significantly contribute to antibiotic resistance in strains, but chromosomally integrated prophages significantly affected strain pathogenicity.

However, the detection of CRISPR sequences has only been found in a small number of K. pneumoniae, according to a study by Founou and colleagues in 2019. ¹⁸ The Cas proteins associated with CRISPRs play a crucial role in the CRISPR-mediated adaptive immune systems found in bacteria and archaea, particularly the Cas3 helicase. ³⁹ In addition, it is regarded as a bacterial defense mechanism as it protects bacteria against phage and horizontal gene transfer by degrading plasmid DNA. On the other hand, a notable discovery of CRISPR-associated Cas in K. pneumoniae in highly resistant strains, including ST152 and ST607, indicated the likely involvement of CRISPR-associated Cas3 in gaining resistance genes. This phenomenon can be explained by the existence of CRISPR-associated Cas genes, located between genes that code for proteins involved in metabolism and antibiotic resistance. ⁴⁰ Additionally, these two highly resistant strains were discovered to possess numerous phages with the CRISPR-associated cas genes, highlighting the need for further research on the development and spread of antibiotic resistance. ¹⁸

Only one isolate of K. pneumoniae from a single area of Iraq was examined in this study, which can be considered a limitation of this study; larger datasets are required for validation.

Conclusion

The first-ever complete WGS study of the K. pneumoniae ST437 strain, which is currently spreading across Iraq, is presented in this research. Our isolates exhibited antibiotic efflux as their primary mode of antimicrobial resistance, with subsequent mechanisms including drug inactivation, protection of antibiotic targets, alteration of antibiotic targets, and replacement of antibiotic targets. Effective management of antimicrobial resistance requires diligent monitoring and surveillance of the prevalence of resistant bacteria, along with identification and tracking of resistance genes and their specific locations. Implementing these strategies can restrict the occurrence of novel and diverse AMR genes, which have the potential to lead to new human diseases in the future.

Authors’ Contribution

S.A: Study concept and design, data acquisition, writing the manuscript; H.A.S: Study concept and design, reviewing the manuscript; K.A: Data analysis and interpretation; O.K: Study concept and design, drafting and reviewing the manuscript. All authors have reviewed, read, and approved the final manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflict of Interest

None declared.

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