Early Response of Antimicrobial Resistance and Virulence Genes Expression in Classical, Hypervirulent, and Hybrid hvKp-MDR Klebsiella pneumoniae on Antimicrobial Stress

Klebsiella pneumoniae is an increasingly important hospital pathogen. Classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) are two distinct evolutionary genetic lines. The recently ongoing evolution of K. pneumoniae resulted in the generation of hybrid hvKP-MDR strains. K. pneumoniae distinct isolates (n = 70) belonged to 20 sequence types with the prevalence of ST395 (27.1%), ST23 (18.6%), ST147 (15.7%), and ST86 (7.1%), and 17 capsular types with the predominance of K2 (31.4%), K57 (18.6%), K64 (10.0%), K1 (5.7%) were isolated from patients of the Moscow neurosurgery ICU in 2014–2019. The rate of multi-drug resistant (MDR) and carbapenem-resistant phenotypes were 84.3% and 45.7%, respectively. Whole-genome sequencing of five selected strains belonging to cKp (ST395K47 and ST147K64), hvKp (ST86K2), and hvKp-MDR (ST23K1 and ST23K57) revealed blaSHV, blaTEM, blaCTX, blaOXA-48, and blaNDM beta-lactamase genes; acr, oqx, kpn, kde, and kex efflux genes; and K. pneumoniae virulence genes. Selective pressure of 100 mg/L ampicillin or 10 mg/L ceftriaxone induced changes of expression levels for named genes in the strains belonging to cKp, hvKp, and hybrid hvKp-MDR. Obtained results seem to be important for epidemiologists and clinicians for enhancing knowledge about hospital pathogens.


Introduction
Klebsiella pneumoniae is an increasingly important hospital pathogen causing a wide range of infections including urinary tract infections, pneumonia, bacteremia, and liver abscesses. In severe clinical cases, it can also lead to multiple organ failure, or even death [1]. Two different evolutionary genetic lines, classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp), were described and are both global pathogens [2]. Most multidrugresistant (MDR) K. pneumoniae strains belong to particular clones (ST11, ST395, ST147, etc.) producing beta-lactamases in combination with other functional classes of resistance determinants [3]. Hypervirulent K. pneumoniae were attributed to sequence types ST23, ST86, ST65, etc., and capsular types K1, K2, K57, K20, etc. [4]. Virulence determinants of hvKp include siderophore systems for iron acquisition, increased capsule production, and the colibactin toxin commonly located on virulence plasmids [5].
Recent studies have shown the ongoing evolution of K. pneumoniae resulting in the generation of hybrid hvKp-MDR strains. Mechanisms for the emergence of such strains can be a result of acquiring hypervirulent plasmids by cKp [6], acquiring MDR plasmids can be a result of acquiring hypervirulent plasmids by cKp [6], acquiring MDR plasmids by hvKp [7], and acquiring hybrid virulence-MDR plasmids [8]. However, the relative expression of resistance and virulence genes in bacteria of cKp, hvKp, and hybrid hvKp-MDR evolutionary branches is poorly studied.
This study aimed to determine the genetic lines of K. pneumoniae strains collected in Moscow neurosurgery ICU in 2014-2019, to identify resistance and virulence genes in their cells, and to estimate relative expression levels of such genes in selected strains belonging to epidemiology significant genetic lines cKp (ST395 K47 and ST147 K64 ), hvKp (ST86 K2 ), and hybrid hvKp-MDR (ST23 K1 and ST23 K57 ).

Bacterial Isolates and Clinical Data
K. pneumoniae caused about 28% among the agents of nosocomial infections in neurosurgery ICU during the period from January 2014 to May 2019. The incidence rates of K. pneumoniae infections were 8.0 per 100 patient infections of the central nervous system, 4.3/100 of bloodstream infections, 26.3/100 of respiratory infections, and 25.3/100 of urinary tract infections [9,10]. A total of 545 K. pneumoniae clinical isolates were collected from 283 patients in this period, including those isolated from the respiratory system (n = 271), urine (n = 166), the nervous system (n = 41), blood (n = 36), surgical wounds (n = 27), and other (n = 4).

K. pneumoniae Sequence Types and Capsular Types
Seventy non-duplicate isolates selected from the collection of 545 isolates were characterized by sequence types and capsular types. These isolates were collected from the respiratory system (n = 34), urine (n = 19), the nervous system (n = 8), blood (n = 6), and surgical wounds (n = 3). As a result, 20 sequence types were identified, the majority of them were ST395, ST23, ST147, and ST86, and a total of 17 capsular types were identified. Predominant K-types were K2, K57, K64, and K1 (Figure 1).

Relative mRNA Levels of Antimicrobial Resistance and Virulence Genes in K. pneumoniae Cells
The expression levels of the resistance genes in all K. pneumoniae strains during growth without selective pressure of antimicrobials were different: the chromosomal beta-lactamase genes bla SHV were transcribed significantly lower compared with those of the reference gene rpoD. In contrast, other beta-lactamase genes (bla TEM, bla CTX-M , bla OXA-48 , and bla NDM ), as well as porin gene ompK36 were transcribed at higher levels compared with the reference gene, with the exception of bla TEM in the hvKp-MDR strain of ST23 K1 which exhibited lower expression. The efflux pump genes and the virulence genes were expressed mostly at the same or lower levels compared with the rpoD gene, with the exception of two virulence genes: treC in the strains of ST395 K47 , ST147 K64 , ST23 K1 , and ST23 K57 ; and celB in the strain of ST86 K2 (Figure 3).

Discussion
K. pneumoniae was one of the major nosocomial pathogens in a Moscow neurosurgery ICU during the period from January 2014 to May 2019, causing about 28% infections including those of the central nervous system, bloodstream, respiratory tract, and urinary tract. This rate was similar to those in the Multispecialty Hospital, Riga, Latvia in 2017-2020 (16-20%) [11], and significantly lower than those reported from 15 China centers in 2012-2016 (52.4%) [12].
Non-duplicate 70 K. pneumoniae isolates collected from 62 patients were attributed to specific genetic lines, virulence, and antimicrobial resistance genotypes. Single isolates were collected from 54 patients and two isolates from eight patients. Double isolates were studied from one patient in a case of their differences in ST (Patients 19, 32, 36, 46, and 48), K-type (Patient 35), and antimicrobial resistance genes profiles (Patients 4 and 39) (Table  S1). Two isolates collected from the trachea and urine of the Patient 4 attributed to ST147 K64 carried (bla TEM +bla SHV +bla CTX-M +bla OXA-48 +bla NDM ) and (bla SHV +bla OXA-48 ) beta-lactamase genes, respectively. Two isolates obtained from the trachea of Patient 39 belonged to ST23 K57 (bla TEM +bla SHV +bla CTX-M ) and (bla SHV +bla CTX-M +bla OXA-48 ) beta-lactamase genes, respectively. Different antimicrobial resistance gene profiles of K. pneumoniae named isolates possibly indicate the evolution events in the patient's body as described previously [13].
Five K. pneumoniae strains belonging to prevalent STs and K-types (cKp, ST395 K47 , and ST147 K64 ; hvKp, ST86 K2 ; and hybrid hvKp-MDR, ST23 K1 , and ST23 K57 ) were selected for further comparative study of whole-genome sequences, antimicrobial resistance phenotypes, hypermucoviscosity, and altered expression levels of virulence and resistance genes in response to beta-lactams effect (AMP and CRO). It was shown that K. pneumoniae isolates belonging to ST86 K2 , ST23 K1 , and ST23 K57 demonstrated hypermucoviscosity phenotype in contrast with isolates of ST395 K47 and ST147 K64 , which confirmed the virulent phenotype of three isolates. All K. pneumoniae isolates carried bla SHV genes, including extended-spectrum beta-lactamase (ESBL) variant bla SHV-12 , broad-spectrum variants bla SHV-28 and bla SHV-33 , and narrow-spectrum variants bla SHV-67 and bla  . These alleles of bla SHV genes were previously described in Portugal, Turkey, China, Russia, and Spain [22,35,36]. Three K. pneumoniae isolates (ST395 K47 , ST147 K64 , and ST23 K1 ) carried the bla TEM-1B , bla CTX-M-15 , and bla OXA-1 genes. It was reported that these genes were horizontally transferred by the IncFIA-FIB-FII and IncHI2 plasmids [37,38]. One isolate (ST23 K57 ) carried the bla CTX-M-55 and bla OXA-1 genes; such gene combination was reported previously from China [39]. It should be noted that MDR isolates of ST395 K47 and ST147 K64 additionally carried two carbapenemase genes, i.e., bla OXA-48 and bla NDM-1 . Previously, it was reported that K. pneumoniae clinical isolates of ST395 and ST147 harbored bla NDM-5 and bla OXA-181/232 in Nepal and that ST11 harbored bla NDM-1 and bla OXA-48 in Greece [33,40]. Of greatest interest are K. pneumoniae isolates belonging to the hvKp evolutionary branch, which acquired the resistance genes and became a hybrid hvKp-MDR. In our study, a hvKp-MDR isolate of ST23 K1 carried simultaneously bla CTX-M-15 and bla OXA-48 genes, similar to a recently published study [34]. Moreover, another hvKp-MDR isolate of ST23 K57 carried not only the bla CTX-M-55 and bla OXA-48 genes but additionally the bla NDM-1 gene. This is the first report describing K. pneumoniae of ST23 K57 genetic-line-acquired cefalosporinase gene bla CTX-M-55 , and two carbapenemase genes bla OXA-48 and bla NDM-1 , which is particularly alarming. The incidence of high-risk clone ST383 carrying bla CTX-M-14b gene and two carbapenemase genes bla NDM-1 and bla OXA-48 combining both resistance and virulence elements was recently published [8] as well as the incidence of ST147-carrying bla CTX-M-15 , bla NDM , and bla OXA-181 genes [41]. Additionally, we detected the efflux pump genes (acrA, acrB, oqxA, oqxB, kpnE, kpnF, kdeA, and kexD) because of their clinical significance for K. pneumoniae beta-lactam resistance presented recently [41]. In our study, one strain of ST23 K1 carried four efflux pump genes: acr and oqx of RND-type systems, kde of MATE-type, and kpn of SMF-type. The rest of the four strains carried five efflux pumps: acr, oqx, kde, kpn, and additionally the kex gene of the RND-type efflux system. The same efflux pump genes were detected recently in K. pneumoniae clinical isolates, which exhibited co-resistance to beta-lactams and aminoglycosides, glycopeptides, fluoroquinolones, and tetracyclines in India [41]. Interestingly, the efflux pumps were associated with bacterial virulence, namely biofilm formation [42].
It is known that multiple biomarkers have been shown to predict hvKp isolates: peg-344, iroB, iucA, plasmid-encoded rmpA, and rmpA2 and quantitative siderophore production (entB and ybtS) [43]. In this study, these genes were detected only in the genomes of hvKp isolates of ST86 K2 , ST23 K1 , and ST23 K57 . In contrast, K. pneumoniae virulence genes (uge, wabG, fimH, mrk, treC, cellB, and ureA), common for both hvKp and cKp, were detected in all five isolates. This is in agreement with recently published data [44]. Two strains of ST23 K1 and ST23 K57 are characterized as hybrid hvKp-MDR. Thus, the data obtained in this study indicate the ongoing formation of hybrid K. pneumoniae on the base of the ST23 genetic line, which was already defined in the last decade [7,22,35,45].
We estimated the basal expression levels of K. pneumoniae resistance and virulence genes at non-selective conditions in vitro and the fold change of expression levels in presence of AMP and CRO. It was shown that ESBL gene bla SHV-12 expressed~4-fold higher in the MDR strain of ST395 K47 than bla SHV -type genes coding broad-spectrum and narrowspectrum beta-lactamases in the remaining K. pneumoniae strains. This is in agreement with previously reported data that ESBL variants of the bla SHV-12 gene expressed higher than non-ESBL variants [46]. These genes did not change their expression after 90 min growing at 100 mg/L AMP or 10 mg/L CRO.
Beta-lactamase genes bla TEM, bla CTX-M , bla OXA-48 , and bla NDM and porin gene ompK36 were expressed at higher levels, with the exception of bla TEM in the hvKp-MDR strain of ST23 K1 . Notably, the expression levels of the beta-lactamase (bla TEM and bla CTX-M ) and carbapenemase (bla OXA-48 , and bla NDM ) genes were higher in cKp-MDR strains than those in hybrid hvKp-MDR strains. Possibly, the reason for this observation is the higher metabolic load in Klebsiella cells producing resistance and virulence factors simultaneously. The basal level of efflux pump gene expression was very different for cKp, hvKp, and hybrid hvKp-MDR strains. In cKp-MDR strains, 5-6 efflux genes were expressed on the same level as reference gene ropD, and 2-3 genes were lower than the reference. In the hvKp strain, one efflux gene expression was higher, three genes were expressed at the same level, and four genes were lower than the reference gene. In contrast, major efflux genes in hybrid hvKp-MDR strains were expressed lower than the rpoD gene, and one gene in the strain of ST23 K57 was expressed on the same level as the reference. Interestingly, the previously described expression of the arcB efflux gene showed upregulation of this gene in carbapenem-resistant K. pneumoniae strains compared with non-resistant ones [47]. The relatively high expression level of efflux genes in cKp and hvKp strains indicates the importance of efflux pumps for virulence of both Klebsiella evolutionary branches that are consistent with previous reports [48]. It is known that efflux pumps use different antimicrobials as substrates. Our results suggest that the change in efflux gene expression in certain K. pneumoniae genetic lines may reflect differences in bacterial surface structures in particular K-types: downregulation in the strains of K47 and K2, and upregulation in the strains of K1 and K57 in response to AMP; and upregulation in the strains of K64 and K57 in response to CRO (Figure 4).
It was shown in our study that transcripts of K. pneumoniae virulence genes common for both the cKp and hvKp evolutionary branches (uge, wabG, fimH, and treC) were present at higher levels in the cKp strains of ST395 K47 , ST147 K64 , and hvKP ST86 K2 than in the hybrid hvKp-MDR strains of ST23 K1 and ST23 K57 . In conditions containing 100 mg/L AMP, these genes were upregulated in cKp and hybrid hvKp-MDR strains (fimH 2.3-4.6-fold) and wabG in hvKp-MDR strains (6.1-14.9-fold), while downregulated in the hvKp strain (uge, wabG, and treC, 3.2-4.9-fold). In conditions with 10 mg/L CRO, only the wabG gene was upregulated in hybrid hvKp-MDR strains (2.3-14.9-fold). The expression levels of the remaining virulence genes common for cKp and hvKp (celB and ureA), as well as common for only hvKp (rmpA, iroN, iroD, and allR), were approximately equal in all studied strains at non-selective conditions. The celB gene expression at AMP medium was upregulated in hvKp-MDR strains (3.7-8.0fold); the ureA gene expression was upregulated in the cKp strain of ST147 K64 (13.9-fold) and hvKp-MDR strains of ST23 K1 (9.2-fold) and ST23 K57 (3.2-fold). It was detected that CRO induced upregulation of the ureA gene in a hvKp-MDR strain of ST23 K57 (4.3-fold) and downregulation of this gene in a hvKp-MDR strain of ST23 K1 (2.6-fold). Expression levels of virulence genes common for hvKp strains were not changed under selective pressure generated by AMP or CRO in the growth media ( Figure 4).
In summary, expression levels of antimicrobial resistance and virulence genes were characteristic for K. pneumoniae of different certain genetic lines. Selective pressure by sub-inhibitory concentrations of ampicillin or ceftriaxone induced differential upregulation or downregulation of these genes depending on the strain belonging to classical cKp, hypervirulent hvKp, or hybrid hvKp-MDR evolutionary branches. Results obtained in this study may be fruitful for future studies of evolution, the spread of antimicrobial and virulence genetic determinants, and the clinical impact of K. pneumoniae genetic lines.

Bioethical Requirements and Patients
K. pneumoniae isolates were collected from the patients of the neuro-intensive care unit (Neuro-ICU) in a specialized Neurosurgical Hospital in Moscow, Russia. Following the requirements of the Russian Federation Bioethical Committee, each patient signed informed voluntary consent to treatment and laboratory examination. The materials used in the study did not contain the personal data of patients.

Bacterial Isolates, Identification, and Growth Conditions
Seventy K. pneumoniae isolates were collected from the respiratory system, blood, urine, cerebrospinal fluid, and wounds of 62 patients of the neuro-ICU. Bacteria identification was performed using by a Vitek-2 Compact instrument (BioMérieux, Paris, France) and a MALDI-TOF Biotyper (Bruker Daltonics, Bremen, Germany). Bacteria were grown at 37 • C with agitation on Luria-Bertani broth (Difco Laboratories, Detroit, MI, USA) and Muller-Hinton broth (Becton Dickinson, Franklin Lakes, NJ, USA). Bacterial isolates were stored in 20% glycerol at minus 80 • C.

Hypermucoviscousity Testing
The string test was used for the identification of hypermucoviscous K. pneumoniae strains growing on the plates with Luria-Bertani broth (Difco Laboratories, Detroit, MI, USA) overnight at 37 • C [23]. The positive test was assigned if a colony of K. pneumoniae formed viscous strings >5 mm length using a standard bacteriological loop.

K. pneumoniae Sequence Type and Capsular Type Identification
Sequence types (STs) of K. pneumoniae isolates were determined by the Multilocus Sequence Typing (MLST) scheme of Pasteur Institute (Paris, France) using the previously published primers [50,51]. The PCR capsular serotyping of the K. pneumoniae isolates was performed using specific primers for the wzy gene associated with K serotypes K1, K2, K20, and K57 [52] and by wzi gene sequencing for identification of capsular types K23, K27, K28, K31, K47, K60, K62, and K64 [53]. Bacterial thermolysates were used as DNA templates for amplification.

cDNA Synthesis and Quantitative Real-Time PCR
One µg of isolated total RNA was used for cDNA synthesis with RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using qPCRmix-HS SYBR (Evrogen, Moscow, Russia) and the CFX96 Real-Time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) with the following program: 40 cycles of 20 s at 95 • C for denaturation, 20 s at 61 • C for annealing, 30 s at 72 • C for extension and SYBR Green detection. The melting curve analysis in the temperature range from 60 • C to 94 • C, with a fluorescence estimation step of 0.2 • C, was performed to confirm the specificity of the reaction. Relative quantification of the target gene expression was normalized with reference genes proC, recA, and rpoD expression. Three technical replicates per each of the three biological samples were used for statistical validity. The relative transcript levels of antimicrobial resistance and virulence genes were calculated using the 2 −∆∆Ct method [56]. A heat map of changes in gene expression levels relative to reference genes was designed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com accessed on 1 November 2021). Gene expression levels of each gene at present of AMP and CRO were compared to those in conditions without antimicrobials.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antibiotics11010007/s1, Table S1: Clinical characteristics, susceptibility to antibacterials, and antimicrobial resistance gene profiles of K. pneumoniae clinical isolates collected from 62 patients of Moscow Neuro-ICU, Table S2