Molecular characterization of the whole genome in clinical multidrug-resistant strains of Klebsiella pneumoniae

Introduction: Antimicrobial resistance (AMR) is a major public health concern. The spread of AMR-encoding genes between enterobacteria, especially in Klebsiella pneumoniae strains, lead to failure in the treatment of most individuals. The aim of this study was to characterize multi-drug resistant (MDR) clinical K. pneumoniae isolates that produce extended-spectrum β -lactamases (ESBLs) from Algeria. Methodology: The isolates were identified using biochemical tests, and the identification was confirmed by mass spectrometry using VITEK® MS (BioMerieux, Marcy l’Etoile, France). Antibiotic susceptibility testing was assessed by the disk diffusion method. Molecular characterization was performed by whole genome sequencing (WGS) using Illumina technology. Sequenced raw reads were processed using bioinformatics parameters: FastQC, ARIBA, and Shovill-Spades. Multilocus sequence typing (MLST) was used to estimate the evolutionary relationship between isolate strains. Results: Molecular analysis resulted in the first detection of blaNDM-5 encoding K. pneumoniae in Algeria. Other resistance genes were blaTEM , blaSHV , blaCTX -M, aac(6')-Ib-cr , qnrB1 , qnrB4 , qnrB19 , qnrS1 , gyrA and parC variants. Conclusions: Our data demonstrated a very high level of resistance in clinical K. pneumoniae strains which were resistant to most common antibiotic families. This was the first detection of K. pneumoniae with the blaNDM-5 gene in Algeria. Surveillance of antibiotic use and measures for control should be implemented to reduce occurrence of AMR in clinical bacteria.


Introduction
Prolonged exposure of Enterobacteriaceae to antibiotics and the selective pressure resulting from the excessive, clinically unnecessary, use of antibiotics contributes to the emergence of antibiotic resistance (AMR) that is a growing global health concern.The spread of infections resistant to the currently available antibiotics could contribute to a death every three seconds globally by 2050 [1].Currently patients with infections caused by antibiotic-resistant bacteria are at high risk of long hospital stays, treatment failure, high mortality, and high health care costs [2].
The prevalence of multi drug resistant (MDR) Enterobacteriaceae, especially those which are resistant to β-lactams, has been increasing worldwide because these antibiotics are important therapeutic choices for treating infections in humans [3].
K. pneumoniae is a notorious bacterium, and by acquiring additional antimicrobial resistance, it could be the origin of increasing nosocomial infections.This species is known to be prone to MDR and hypervirulent strain emergence [18].
K. pneumoniae cause severe infections including liver abscess, pneumonia, and sepsis by the production of enzymes such as extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC β-lactamases (pAmpC) that are enzymes commonly isolated from Enterobacteriaceae [4].Nowadays, enterobacteria producing ESBLs and β-lactamase enzymes are frequently isolated from nosocomial and communityacquired infections [5].Therefore, acquired carbapenemase-encoding genes in enterobacteria constitute a real clinical concern for antimicrobial management.ESBL, pAmpC and the carbapenemases are largely produced by MDR enterobacteria such as Klebsiella spp isolates.The corresponding genes are located on plasmids and other mobile genetic elements [6].This is alarming because these plasmids frequently carry genes encoding resistance to other classes of drugs such as aminoglycosides, trimethoprimsulfamethoxazole, fluoroquinolones, and can move horizontally among bacteria [7].This is especially the case with Klebsiella spp which is one of the bacteria in need of development of new therapeutic compounds [8].The objective of this study was to evaluate the level of antibiotic resistance and to perform the molecular characterization of clinical K. pneumoniae strains isolated from out-patients and in-patients admitted to different wards of the hospital in Oum El Bouaghi, Algeria.

Samples collection
30 Bacterial isolates were provided by the laboratory of Microbiology of the hospital at Oum El Bouaghi, Algeria.They were isolated between December 2021-February 2022 from various clinical samples (urinary tract infections, hemoculture and pus) collected from out-patients and in-patients admitted to different wards of the hospital.
The isolates were grown on MacConkey agar (BioMerieux, Marcy l'Etoile, France) for 24 hrs at 37 ± 1 °C.The isolates were identified using biochemical tests, and the identification was confirmed by mass spectrometry using VITEK® MS (BioMerieux, Marcy l'Etoile, France).VITEK® MS PRIME is a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) mass spectrometer.The instrument analyzes material from microbial cultures to identify the microorganism.The samples were exposed to multiple laser shots inside VITEK® MS PRIME [1], and this was designed to incorporate additional benefits and enhance the use of MALDI-TOF technology [1].
The screening of ESBL production was performed by the double-disc synergy test (DDST) between clavulanic acid and third generation cephalosporins (cefotaxime, ceftazidime, aztreonam and cefepime) [9].The test was considered positive when a "champagne cork" aspect was observed.

DNA sequencing
Genomic DNA was extracted using a QIAxtractor (Qiagen, Valencia, CA), and library preparation was performed by using a Nextera XT DNA sample preparation kit (Illumina, San Diego, CA) according to the Illumina protocol.The libraries were sequenced with the Illumina MiSeq platform (2x 300-bp pairedend reads) with a minimum of mapped-reads depth of 40-fold.
Contaminant searches and molecular identification were performed for each sample using the centrifuge.This method performs abundance analyses at all taxonomic levels (e.g.strain, species, genus) [10].Quality control metrics were examined across the whole collection as a batch report to ensure a mean read base-pair quality score of Q ≥ 20 and a read length of 70% of the original read length.Quality-filtered Illumina reads were assembled using Unicycler [11].Contigs were annotated using Bakta.

Gene annotation
To define the presence of specific genes and their alleles, we used ARIBA [12], DIAMOND [13], and the following databases: Multilocus sequence typing MLST database, serotypeFinder O:H typing database, the fimH typing database, and curated databases of AMR genes/Single Nucleotide Polymorphism (SNPs) including Res-Finder, NDARO, and CARD.

Multilocus sequence typing (MLST) and clonality analysis
According to data of MLST, all isolates belonged to the sequence type ST307.Clonality analysis performed by cgMLST on mean 2145635 bases (https://microreact.org/project/gekjDYwFLTkanVvTG Bzwoi-confile) showed that the most similar isolates 03 and 13 (ST307) differ by 39 SNPs.The isolates did not therefore emerge as a recent clonal spread but probably correspond to an endemic reservioir of ST307.

Discussion
The World Health Organization (WHO) has recommended that countries should develop AMR surveillance programs to collect and integrate antimicrobial use.
Our genomic data analysis, presented in Table 1, confirmed that all K. pneumoniae strains (n = 4) are ESBLs.A co-existence of three ESBL genes (CTX-M, TEM, and SHV) was found in two strains (S03 and S13), and this was already reported in Algeria [19], and in other countries [20].Association of ESBL genes lead to selection of resistance genes in hospitals as well as in other sectors.In addition, the presence of multiple βlactamase genes with several variants in K. pneumoniae isolates would also be the cause of MDR and other extremely drug resistant (XDR) strains emergence [21].These may lead to high human mortality rates when it comes to clinical infections due to failures of antimicrobial therapies [22].Both blaSHV and blaCTX-M genes are present in the two strains (S01 and S09) analyzed in K. pneumoniae.The coexistence of blaSHV and blaCTX-M genes was also observed in previous studies conducted in Algeria [23].
Through this investigation, the CTX-M genes have been identified in all K. pneumoniae (S01, S03, S09, and S13); blaCTX-M-15 type variant (CTX-M-1 group) in particular is a well distributed strain throughout the world, including Algeria [24].
A blaSHV-11 gene was found in S01 and S09 strains that procures a broad-spectrum resistance to antibiotics in the K. pneumoniae strain.Two strains (S03 and S13) harboring the blaSHV28 and the blaTEM-1 genes have been identified, and have already been a subject of study in Algeria [23].No data was available on the functional aspect of variant gene blaTEM-1.Hyperproduction of TEM-1 β-lactamase mediated by the promoter Pa/Pb which was detected in two K. pneumoniae strains (S03 and S13) has never been addressed in Algeria, and according to other studies blaTEM-1(PaPb) is only responsible of high resistance to piperacillin-tazobactam (TZP) [25].
The OXA gene was found present in three strains of K. pneumoniae.The OXA-1 gene was also detected in Algeria [26].OXA-1 variant gene was discovered in coexistence with ESBL genes.This result is consistent with previous studies that showed that these genes are commonly present in combination with CTX-M-15, SHV-1, TEM-1 β-lactamases, PMQR (plasmid mediated quinolones resistance) determinants, and aac(6')-Ib-cr in a community of K. pneumoniae strain [27].
Previous studies have demonstrated that NDM-5 offer greater resistance than NDM-1 genes [34].From what we observed, the detection of New Delhi Metalloβ-lactamases genes of type NDM-5 in K. pneumoniae (S09) strains is considered as a first in Algeria, However the blaNDM-5 gene has been reported only in other clinical isolates such as Acinetobacter baumannii [28], Escherichia coli [29], and Enterobacter cloacae [30].
In contrast, in other countries the blaNDM-5 gene has been identified mainly in clinical Enterobacteriaceae.The carbapenems represent the latest threat to public health especially in the case of nosocomial transmission of the blaNDM gene which has occurred in many countries [31].However, in Algeria, NDM-5 gene was detected in K. pneumoniae from animal origins such as white storks [32].This finding confirms that wildlife in Algeria could serve as reservoirs of MDR K. pneumoniae, in addition to its presence in fecal samples of companion animals [33].One finding of the present study is the existence of fluoroquinolone-resistant strain of K. pneumoniae carrying two plasmid mechanisms of resistance to quinolones: the aac(6')-Ib-cr variant gene harbored by two strains (S01 and S13) and the qnrB1 gene present in three strains (S01, S03, and S13); these were also previously identified in Algeria [35].One strain (S09) had aquired the qnrS1 gene.This is the second report of qnrS1 in ESBL-K.pneumoniae in Algeria [26].It was previously described that qnrB was predominant among strains from Africa that qnrS.An association has been found between the production of blaCTX-M-15, qnrB1 and/or qnrS1 gene in all strains of K. pneumoniae, based on data from studies in Algeria [36] and in other countries [37] which report cases of K. pneumoniae harboring blaCTX-M-15 and aac(6')-Ib-cr [38].
Based on studies conducted in Algeria, we report for the first time the chromosomal mechanism of resistance to quinolones in three K. pneumoniae strains (S01, S03, and S13) mediated by the Ser83Leu (S83I) substitution in gyrA gene, which is consistent with previous studies from several countries [39].The gyrA (S83I) gene has been identified in strains of K. pneumoniae carrying plasmid mechanisms especially the qnrB1 gene.Thus, the appearance of chromosomal mutations in the genes encoding DNA gyrase can increase the level of resistance of the bacteria to quinolones [40].
Our qnr positive strains show resistance to aminoglycosides, cotrimoxazole, sulfonamide trimethoprime, fosfomycin, and cycline.This could be explained by the fact that the plasmid support of quinolone resistance is present in a cassette as part of an integron carrying the other PMQR genes like blaCTXM-15, qnrB and the multiresistance genes to other antibiotics [41].
Few studies in Algeria have identified the detailed expression of resistance to aminoglycosides.Our data show that all the strains were resistant to aminoglycosides but with different gene expression levels, and this includes aph( 6)-Id, aac(3)-IIe, aac(6')-Ib-cr, aph(3')-Ia, and aph(3'')-Ib genes.One strain (S01) of K. pneumoniae harbored genes of the type aadA11, and this was first detected in Algeria from clinical strains of K. pneumoniae.Among these identified genes, the most concerning is the aac(6')-Ibcr variant gene [42].All of aminoglycosides resistant genes discovered in this study have not been studied in Algeria, especially since most of them have been located in mobile elements (plasmids, integron, transposon and integrative conjugative element) [43].
This study is the first investigation in Algeria analyzing resistance to sulfonamides and tetracycline in clinical strains of K. pneumoniae.Resistance to sulfonamides in all K. pneumoniae strains (n = 4) is due to sul1 and sul2 genes.In addition, we identified resistance to trimethoprime by dfrA1, A7, dfrA14, and dfrB1.All the strains showed resistance to fosfomycine and carried a fosA gene.There were three strains that carried tetA, tetG, and tetR genes that lead to resistance to cyclines.The acquisition of resistance to fosfomycin and tetracycline by clinical isolates of K. pneumoniae sharing ESBL and/or carbapenemases represents a new threat that complicates the situation and shows that K. pneumoniae remains a very serious causative agent of therapy failure [44].
The most similar isolates S03 and S13 (ST307) differed only by 39 SNPs and both carried the ESBLs genes: blaTEM-1, blaCTX-M-15, blaOXA-1, blaSHV-28, and blaTEM (PaPb).They belong to the same strain ST-307 as indicated in Figure 1 that demonstrates the phylogenetic tree of multilocus sequence typing (MLST) of K pneumoniae.The ST307 clone has been previously detected in Algeria by [45].Our results show that this clone was associated with three ESBLs (blaCTX-M-15), qnrB and other genes.Several reports have showed that the origin of MDR ST307 K. pneumoniae was from clinical samples, as well as from other sources [57].The WHO recently declared that the ST307 strain poses critical threat to public health It has also been concluded that ST307 K. pneumoniae can often carry transferable resistance-conferring genes against carbapenems and a variety of additional resistance and virulence determinants, in addition to integrative and conjugative elements, and phages.As a result, ST307 K. pneumoniae is emerging globally as an important vehicle for the dissemination of AMR determinants [58], and has been responsible for several global nosocomial and long-term care center outbreaks [59].The MLST analysis indicated that ST307 consisted of one deep-branching lineage which contained the gyrA S83I mutation in the quinolone resistance determinant region (QRDR) that had global distribution [60].

Conclusions
The present study reports the first analysis and identification of the bacterial resistome of MDR clinical strains belonging to Klebsiella pneumoniae in Oum Bouaghi, Algeria.Our data demonstrate multiple clinical K. pneumoniae strains resistant to most common antibiotic families.This is also the first reported detection of K. pneumoniae producing blaNDM-5 gene in Algeria.Control of antimicrobial resistance requires monitoring and surveillance of the level of emergence of resistant bacteria, as well as the resistance genes and their location.These measures can limit the presence of new diverse AMR encoding genes that could be a source of emergence of new human pathogens in the future.

Table 1 .
Antibiotic resistance-encoding genes found in clinical K. pneumoniae strains.