Multidrug-Resistant Klebsiella pneumoniae Causing Severe Infections in the Neuro-ICU

The purpose of this study was the identification of genetic lineages and antimicrobial resistance (AMR) and virulence genes in Klebsiella pneumoniae isolates associated with severe infections in the neuro-ICU. Susceptibility to antimicrobials was determined using the Vitek-2 instrument. AMR and virulence genes, sequence types (STs), and capsular types were identified by PCR. Whole-genome sequencing was conducted on the Illumina MiSeq platform. It was shown that K. pneumoniae isolates of ST14K2, ST23K57, ST39K23, ST76K23, ST86K2, ST218K57, ST219KL125/114, ST268K20, and ST2674K47 caused severe systemic infections, including ST14K2, ST39K23, and ST268K20 that were associated with fatal incomes. Moreover, eight isolates of ST395K2 and ST307KL102/149/155 were associated with manifestations of vasculitis and microcirculation disorders. Another 12 K. pneumoniae isolates of ST395K2,KL39, ST307KL102/149/155, and ST147K14/64 were collected from patients without severe systemic infections. Major isolates (n = 38) were XDR and MDR. Beta-lactamase genes were identified: blaSHV (n = 41), blaCTX-M (n = 28), blaTEM (n = 21), blaOXA-48 (n = 21), blaNDM (n = 1), and blaKPC (n = 1). The prevalent virulence genes were wabG (n = 41), fimH (n = 41), allS (n = 41), and uge (n = 34), and rarer, detected only in the genomes of the isolates causing severe systemic infections—rmpA (n = 8), kfu (n = 6), iroN (n = 5), and iroD (n = 5) indicating high potential of the isolates for hypervirulence.


Introduction
Healthcare-associated infections (HAI) have posed a huge medical burden to public health worldwide. Klebsiella pneumoniae is one of the clinically significant nosocomial pathogens causing broad spectra of diseases and showing increasingly frequent acquisition of resistance to antibiotics including in intensive care units (ICU) [1]. Today, according to the BIGSDB Institute Pasteur database (https://bigsdb.pasteur.fr/ access date 8 August 2021), 5797 K. pneumoniae sequence types and 711 capsular types have been discovered.

Patients and Bacterial Strains
During the period from Oct. 2017 to Jan. 2019, the following incidence of infections was detected in neurosurgery ICU: 8.4 infections of the central nervous system per 100 patients, 2.7/100 of bloodstream infections, 26.3/100 of ventilator-associated pneumonia, and 23.6/100 of urinary tract infections. K. pneumoniae accounted for 33, 31, 23, and 25% among the agents of named infections, correspondingly [3]. The subject of this study was 41 resistant-to-antimicrobials K. pneumoniae isolates collected from 20 patients with severe postoperative infections (Table 1).   of Group A were associated with manifestations of vasculitis and microcirculation disorders (patients A, I, and F). Major isolates of Group A (17/29) were collected from the blood and cerebrospinal fluid, less (7/29)-from the endotracheal aspirate.
Group B consists of 12 strains isolated from the patients without increasing markers of systemic inflammation and septic reaction. Major isolates of Group B were from the endotracheal aspirate and urine (10/12) and the rest from the nervous system. One patient in this group died due to the underlying disease, videlicet multiple metastases of kidney cancer in the brain (Tables 1 and 2). It should be noted that isolates of Group A were obtained from 2-3 body sites of one patient: three isolates-from blood, urine, and cerebrospinal fluid of the patient D; three isolates-from endotracheal aspirate and cerebrospinal fluid of patient S; and three isolates-from blood and cerebrospinal fluid of the patient L ( Table 2).

Phylogenetic Analysis
The phylogenetic tree was constructed on the base of combined gene sequences of MLST profiles; two clusters were revealed. Cluster I consisted of one ST147 referring to one isolate not associated with severe manifestations of systemic infections (Group B). Cluster II included two sub-clusters: IIa consisting of one ST86 associated with a pronounced systemic inflammatory response (Group A); IIb consisting of 10 genetic lineages including 8 sequence types (ST14, ST23, ST39, ST76, ST218, ST219, ST268, and ST2674) referred to the strains of Group A, and 2 sequence types (ST307 and ST396) including isolates both Group A and Group B (Figure 2). . It was interesting in that K. pneumoniae isolates of capsular type K2 belonged to three sequence types (ST14, ST86, and ST395), isolates of capsular type K23-to two sequence types (ST39 and ST76), and isolates of capsular type K57-to two sequence types (ST23 and ST218). The remaining capsular types were associated with only one sequence type: K20-ST268, KL39-ST395, K47-ST2674, K14/64-ST147, K102/149/155-ST307, and K125/114-ST219 (Table 3).

Phylogenetic Analysis
The phylogenetic tree was constructed on the base of combined gene sequences of MLST profiles; two clusters were revealed. Cluster I consisted of one ST147 referring to one isolate not associated with severe manifestations of systemic infections (Group B). Cluster II included two sub-clusters: IIa consisting of one ST86 associated with a pronounced systemic inflammatory response (Group A); IIb consisting of 10 genetic lineages including 8 sequence types (ST14, ST23, ST39, ST76, ST218, ST219, ST268, and ST2674) referred to the strains of Group A, and 2 sequence types (ST307 and ST396) including isolates both Group A and Group B (Figure 2).

Figure 2.
Phylogenetic tree of K. pneumoniae sequence types identified in the study generated by using a web resource NCBI "Blastn" and "Blast Tree View", based on combined gene sequences of MLST profiles.

Whole-Genome Sequencing
Whole-genome sequencing was done for nine isolates including eight isolates of Group A belonging to sequence types/capsular types ST2674/K47, ST23/K57, ST39/K23, ST219/K125, ST218/K57, ST76/K23, ST86/K2, and ST307/K102 and one isolate of Group B belonging to ST395/K39. From 86 to 164 contigs for each genome were obtained, the ge- Figure 2. Phylogenetic tree of K. pneumoniae sequence types identified in the study generated by using a web resource NCBI "Blastn" and "Blast Tree View", based on combined gene sequences of MLST profiles.
Analysis of virulence genetic determinants revealed mrk gene coding type 3 adhesinein all 9 genomes, irp gene of yersiniabactin biosynthesis and ybt gene of yersiniabactin transcriptional regulator-in 7 genomes, fyu gene of siderophore yersiniabactin receptorin 6 genomes, iut gene of ferric aerobactin receptor-in 6 genomes, iuc gene of aerobactin siderophore synthesis-in 5 genomes, kvg gene of capsular polysaccharide synthesis regulator-in 2 genomes. Generally, each genome contained 1-7 virulence genes. Plasmids of eight incompatibility groups (IncC, IncFIA, IncFIB, IncFII, IncHI1B, IncM1, IncM2, and IncR) were identified in the K. pneumoniae isolate genomes, specifically 1-3 plasmids per genome. Molecular systems protecting bacteria from the foreign DNA, Type I Restriction-Modification systems, were detected in four genomes, Type II systems in all nine genomes, but CRISPR-Cas systems were not detected in the genomes (Table 4).

Discussion
K. pneumoniae isolates that caused severe postoperative infections in patients of neuro-ICU between 2017 and 2019 were divided into two groups, A and B, depending on observed clinical manifestations: associated and not associated with the pronounced systemic in-flammatory response. Previously, the association of K. pneumoniae with severe systemic infections was described as the modern trend for ICU; for example, K. pneumoniae were the most frequent infecting species (47%) determined meningitis/encephalitis and 30-day mortality rates, 15% in post-neurosurgical patients [12]. In our study, as in the reports from other countries, the overwhelming majority of K. pneumoniae isolates obtained from neurosurgery patients were MDR and XDR [13]. Moreover, MDR and XDR K. pneumoniae isolates in our study were associated with severe systemic infections, including vasculitis and microcirculation disorders. It was reported previously that K. pneumoniae was associated with leukocytoclastic vasculitis [4][5][6], rapidly progressive retinal vasculitis [14], and acute vasculitis at respiratory infection [15]. In this study, we first identified K. pneumoniae of ST395 and ST307 as bacterial pathogen associated with vasculitis and microcirculation disorders.
A total of twelve sequence types and nine capsular types of K. pneumoniae were identified in this study, which possibly reflects a continuous influx of new genetic lineages into neuro-ICU from other hospitals and regions. The prevalent ST in the ICU was ST395 (16/41 isolates), which is similarly rated to that previously published in the studies from Poland, France, Italy, and Russia [16][17][18][19][20]. Nine STs in our study (ST14, ST23, ST39, ST76, ST86, ST218, ST219, ST268, and ST2674) were identified only for the isolates that caused severe bloodstream infections. Previously K. pneumoniae of ST23, ST86, ST76, and ST218 were described as a hypervirulent pathogen causing bacteremia, sepsis, and liver abscess in India, France, China, Taiwan, and Russia [21][22][23][24][25]. Three STs (ST14, ST39, and ST268) in our study were associated with fatal outcomes. These STs have been reported previously as the agents of severe bloodstream infections in ICU and surgery wards in other countries [26,27]. Two sequence types, ST219, and ST2674 were first identified in this study as the agent of severe sepsis in the patients of ICU. Recently, the ST219 was reported for the environmental MDR K. pneumoniae strains collected from hospital wastewater in Southern Romania [28]; the ST2674 was identified for the environmental isolate from Pakistan (https://bigsdb.pasteur. fr/cgi-bin/bigsdb/bigsdb.pl?page=info&db=pubmlst_klebsiella_isolates&id=5256 access date 8 August 2021).
The high prevalence of polyresistant isolates in our study was associated with antimicrobial resistance genes. The isolates of Group A carried ESBL genes bla CTX-M (22/29), carbapenemase genes bla OXA-48 (12/29), and bla KPC-2 (1/29), which correspond to the reports from Greece and China [27,29,30]. Moreover, four isolates in Group A carrying the only bla SHV were identified (ST218 and ST86). These isolates, as well as four additional isolates of ST268, ST23, and ST76, carried virulence gene rmpA coding regulator of hypermucoid phenotype. It was reported previously that overexpression of rmpA could enhance the virulence of K. pneumoniae isolates in the mouse model [31]. Virulence genes iroN and iroD associated with utilization of trivalent iron were detected in K. pneumoniae of ST23, ST218, ST76, ST86, and ST268 (isolates associated with severe bloodstream infections) which agrees with reports from other countries [22,23,29]. It is known that the majority of pathogenic bacteria including K. pneumoniae possess the iron-acquisition system with a higher affinity for iron than the host, which serves as one of the strategies for increasing bacterial virulence [32]. The kfu gene coding iron uptake system was identified in K. pneumoniae of ST14 and ST219; the latter is a novel genetic lineage carrying the kfu gene [33,34]. Kfu was shown to be a potential virulence factor in the intragastrical murine model, which indicates that kfu might contribute to intestinal colonization [35]. Therefore, the identified virulence genes indicate the high potential of studied isolates for hypervirulence.
The isolates that were not associated with severe manifestations of systemic infections in our study were attributed to ST395, ST307, and ST147. Among them, the isolates of ST395 carrying the bla OXA-48 carbapenemase gene were prevalent. Such K. pneumoniae strains were reported earlier from Hungary and Russia [36,37]. In our study, one isolate of ST147 carried the bla NDM-1 carbapenemase gene, and the same strains have been described worldwide [38,39]. It should be noted that ST307 and ST147 have been estimated as K. pneumoniae High-Risk Clones (HRC) because of worldwide distribution, ability to cause serious infections, and association with polyresistance [40].
The whole-genome study of selected K. pneumoniae isolates belonged to nine sequence types, ST23, ST39, ST76, ST86, ST218, ST219, ST307, ST395, and ST2674, showing the great diversity of these isolates in the combination of virulence genes, antimicrobial resistance genes, heavy metal resistance, and plasmids. Analysis of their resistomes showed that the genes of beta-lactamases bla CTX-M-15 , bla TEM-1B , bla NDM-1 , bla OXA-48 , bla KPC-2 , and bla OXA-1 are represented by a single allele. These alleles were recently reported for K. pneumoniae isolated in Russia [41]. On the contrary, six alleles were identified of bla SHV -type genes, which were not reported in Russia before our study: bla SHV-26 , bla SHV-28 , bla SHV-33 , bla SHV-40 , bla SHV-59 , and bla SHV-182 . In general, all isolates in our work carried bla SHV genes, 68% isolates-bla CTX-M genes, 51%-bla TEM genes, 51%-bla OXA-48 genes, but only one isolate carried bla NDM-1 gene, and only one isolate bla KPC-2 gene. A similar representation of beta-lactamase genes was reported from the European countries and Russia [41,42]. Major isolates in our study were susceptible to amikacin and imipenem, which is consistent with recently published data from Saudi Arabia and Indonesia [43,44]. Interestingly, the rmtB gene encoding 16S rRNA methylase providing resistance to aminoglycosides in the K. pneumoniae ST23 isolate was detected in this study for the first time. This gene was reported earlier for ST258 and ST16 of KPC-producing K. pneumoniae [45]. Moreover, armA gene coding 16S rRNA methyltransferase was detected in K. pneumoniae of ST395 in this study; recently, this gene was detected in K. pneumoniae ST23, ST2502, and ST11 in Italy, Spain, and China, respectively [46,47].
The virulence determinants detected in the genomes were the following: mannoseresistant Klebsiella-like (mrk) hemagglutinin gene critical for K pneumoniae biofilm development in all 9 genomes; aerobactin siderophore locus (iuc, iut) in 6 genomes; regulator of capsular polysaccharide synthesis (kvg) in 2 isolates. A similar distribution of virulence genes was reported recently from Brazil [48]. The yersiniabactin locus (irp, ybt, and fyu) was detected in 7/9 genomes in our study, compared with 40% of K. pneumoniae genomes, particularly amongst those associated with invasive infections [49]. This data confirmed the high virulence potential of the studied clinical isolates. The presence in the genomes of the plasmids of different incompatibility groups indicates a possible role of 'hybrid' plasmids in the formation of K. pneumoniae strains, simultaneously carrying a large number of antibiotic resistance and virulence genes. [9,50].
Future research will focus on studying the structure of plasmids carrying genes for antimicrobial resistance and virulence, as well as the expression of these genes under conditions of different genetic environments and selective pressure of antibiotics. We believe that further study of the microbiology, molecular biology, physiology, and interactions with the host of K. pneumoniae will provide important knowledge to control K. pneumoniae infection in ICUs.

Bioethical Requirements and Patients
Each patient signed informed voluntary consent to treatment and laboratory examination, under the requirements of the Russian Federation Bioethical Committee. The study did not contain personal data of patients; the clinical bacterial isolates information did not include name, date of birth, address, and disease history. The study was a retrospective observational study. The study was done in the neuro-ICU department in a specialized Neurosurgical Hospital in Moscow, Russia, with 300 beds that care for approximately 8000 patients per year, 95% of whom undergo surgery. The surveillance software was designed in-house and integrated into the hospital's electronic health record system [51]. Four types of health-associated infections were surveilled: bloodstream, respiratory and urinary tract infections, and healthcare-associated ventriculitis and meningitis [3].

Whole-Genome Sequencing
Whole-genome sequencing was done on the Illumina MiSeq platform using Nextera DNA Library Preparation Kit (Illumina, Carlsbad, CA, USA) and MiSeq Reagent Kits v3 (Illumina, Carlsbad, CA, United States). The obtained single reads were collected into