Abstract
Aims:
The objective of this study was phenotypic and genotypic characterization of antibacterial-resistant Klebsiella pneumoniae clinical strains isolated in Moscow Transplantology Intensive Care Unit in 2017–2019.
R
Conclusion:
The emergence of novel carbapenemase-producing K. pneumoniae genetic lines in Russia highlights the global negative tendency of multidrug-resistant pathogens spread in high-technological medical centers.
Introduction
Antibacterial resistance (ABR) of human pathogens is a global public health problem, which is cause of death for 50,000 people every year in Europe and the United States. 1 One of the most significant and dangerous agents of drug-resistant hospital infections is Klebsiella pneumoniae, which belonged to ESKAPE group. 2 K. pneumoniae, as other Enterobacterales, is characterized by the combination of their ability to colonize the host organism and the rapid acquisition of resistance to a wide range of antibacterial drugs. 3
The World Health Organization (WHO) has recently published the priority list of three tiers (critical, high, and medium priority) for new antibiotic research and development. Carbapenem-resistant (CR) Enterobacterales including CR K. pneumoniae were attributed to critical-priority bacteria because of empty antibiotic pipeline, high attributable mortality in severe infections, and the dearth of effective infection control measures against these pathogens. 4
K. pneumoniae resistome covers a wide range of genetic ABR determinants that encode molecular mechanisms, among which the main ones are enzymatic inactivation of antibacterials (beta-lactamases, carbapenemases, acetyltransferases, etc.), alteration in the target sites of antibacterials, reduction of permeability, and efflux. 5 The epidemiologically significant ABR genetic determinants of K. pneumoniae are the KPC-, OXA-48, NDM-, and VIM-1-type carbapenemase genes and CTX-M-type extended-spectrum beta-lactamase (ESBL) genes.6–8 Moreover, integrons carrying many different types of gene cassettes (aac, aad, dfr, cml, orf, etc.) were recognized as an important mechanism of multidrug resistance (MDR) and extensive drug resistance (XDR) in K. pneumoniae, providing resistance to aminoglycosides, sulfonamides, fenicoles, and so on. 9
K. pneumoniae is an important MDR human pathogen, one of the main agents of nosocomial infections associated with a great deal of morbidity, mortality, and increased financial burden. In the last two decades, numerous high-risk (HiR) MDR and XDR K. pneumoniae genetic lines have emerged showing superior ability to cause multicontinental outbreaks and continuous global dissemination. 10 Particular attention was focused on the process of intense spread of international K. pneumoniae clones carrying carbapenemase genes. Notably, of all reported outbreaks where K. pneumoniae clonal groups (CGs) were determined, 72% identified one of five common CGs: CG258, CG14/15, CG17/20, CG43, and CG147, despite the fact that more than 150 CGs have already been identified. 11
The purpose of this study was the retrospective phenotypic and molecular genetic characterization of MDR K. pneumoniae clinical strains isolated in the Moscow Transplantology Intensive Care Unit (ICU) and identification of sequence types (STs) among them.
Materials and Methods
Bioethical requirements
The materials used in the study did not contain personal data of patients because the labeling of the clinical isolates did not include name, date of birth, address, disease history, or other personal information. In accordance with the requirements of the Russian Federation Bioethical Committee, each patient signed an agreement with the hospital consenting to treatment and laboratory examination.
Bacterial strains, identification, and growth conditions
Clinical MDR K. pneumoniae strains (n = 63) were isolated from 30 patients of the Moscow Transplantology ICU from December 2017 to January 2019. The inclusion criteria in the study for K. pneumoniae strains were as follows: ABR phenotype, isolation from different patients, different collection date, or different sites of isolation. Strains isolated in the same day from different sites of the patient body or from the environment specimen were marked by (a), (b), or (c) index. Bacterial identification was performed using MicroScan WalkAway 96 Plus (Beckman Coulter, Porterville, CA) and MALDI-TOF Biotyper (Bruker Daltonics, Bremen, Germany) devices. Bacterial cultures were grown at 37°C on Nutrient Medium No. 1 (SRCAMB, Obolensk, Russia) and Muller–Hinton broth (Himedia, Mumbai, India), stored in 20% glycerol at −70°C.
Susceptibility to antibacterial agents, resistance categories
Susceptibility to 26 antibacterials (ABs) of 8 functional groups: penicillins (ampicillin, ampicillin–sulbactam, piperacillin, piperacillin–tazobactam), cephalosporins (cefuroxime, cefoxitin, cefotaxime, ceftriaxone, ceftazidime, cefoperazone–sulbactam, cefepime), carbapenems (ertapenem, imipenem, meropenem, doripenem), aminoglycosides (gentamicin, tobramycin, amikacin), fluoroquinolones (ciprofloxacin, levofloxacin), sulfonamides (trimethoprim, sulfamethoxazole), tetracyclines (tetracycline, tigecycline), and polymyxins (colistin, polymyxin) were determined using a MicroScan WalkAway 96 Plus device (Beckman Coulter) device. The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing, Version 9.0. Escherichia coli strains ATCC 25922 and ATCC 35218 were used for quality control.
The strains were classified as MDR and XDR according to the criteria proposed by German et al. (2018), based on sensitivity to six groups of ABs: aminoglycosides (tobramycin or gentamicin), penicillins (piperacillin–tazobactam), carbapenems (imipenem or meropenem), cephalosporins (cefotaxime, ceftriaxone, or ceftazidime), fluoroquinolones (ciprofloxacin), and sulfonamides (trimethoprim–sulfamethoxazole). Strains resistant to three or four AB groups were classified as MDR, and strains resistant to five or six AB groups were classified as XDR. 12
PCR detection of ABR , virulence, and K serotype-specific genes
Beta-lactamase genes blaCTX-M, blaTEM, blaSHV, blaOXA-48, blaKPC, blaVIM, blaIMP, blaNDM; class 1 and class 2 integrases int1 and int2, gene cassette arrays ins1 and ins2, as well as genes associated with K. pneumoniae virulence rmpA (hypermucoid phenotype regulator), aer (aerobactin), kfu (ferric absorption system), uge (uridine diphosphate-galacturonate-4-epimerase), wabG (glucosyltransferase), fimH (fimbria type I), allS, and allR (allantoin regulon) were detected by PCR using previously described specific primers.13–23 The capsular serotypes of the K. pneumoniae strains were determined by wzi gene sequencing, 24 and allele identification using Institute Pasteur, Paris, France, BIGS database.
Multilocus sequence typing
STs of K. pneumoniae were identified based on allelic profiles of seven housekeeping genes (rpoB, gapA, mdh, pgi, phoE, infB, and tonB), according to the Institute Pasteur, Paris, France, BIGS database protocol.
DNA sequencing and bioinformatics analysis
Sequencing of DNA fragments was carried out using the ABI PRISM BigDye Terminator v.3.1 kit (Applied Biosystems, Foster City, CA). Purified products were analyzed on an ABI PRISM 3100-Avant automated DNA Sequencer at the SINTOL Center for collective use (Moscow, Russia). DNA sequences were analyzed using Vector NTI9 (Invitrogen) and BLAST. Class 1 and class 2 integrons were analyzed using the INTEGRAL database.
The phylogenetic tree of K. pneumoniae STs was constructed using a web resource NCBI “Blastn” and “Blast Tree View”, based on gene sequences of multilocus sequence typing (MLST) profiles. The sequence of the ST147 allelic profile was used as a reference.
Database submissions
Ninety nucleotide sequences were submitted to GenBank database: blaSHV-type genes [MK876287–MK876294, MN652553, MN688550–MN688558], blaTEM-1 genes [MH544753–MH544756, MK113952–MK113955], blaCTX-M-15 genes [MK113956–MK113960, MN688543–MN688549], blaCTX-M-14 gene [MN168268], blaOXA-48 genes [MK867758–MK867763], blaNDM-1 genes [MK917649, MN398861]; gene cassette arrays of class 1 integrons aacA4 [MK649936, MK889497], dfrA5 [MN186299], aadB-aadA1 [MK799964, MK800018, MK889498, MK889500], and dfrA1-orfC [MK649937, MK800019, MK799963, MN186297, MN186300]; wzi genes [MK910971–MK910981, MK910983–MK910993, MN186298, MN191634–MN191637, MN201604, MN201605, MN216142, MN216143].
Information about 63 K. pneumoniae strains was submitted to the Institute Pasteur BIGS database, Paris, France (Table 1).
Clinical Klebsiella pneumoniae Strains, Isolated in Moscow Transplantology Intensive Care Unit
Areas closest to the patient.
ND, K-type is not typable.
(a), (b), and (c): different sites of patient body or environments.
MDR, multidrug resistant; XDR, extensive drug-resistant; ST, sequence type; CG, clonal group; ID, identification number in the BIGS database, Institute Pasteur, Paris, France.
Results
Bacterial strains
A total of 63 clinical K. pneumoniae strains were collected from 30 patients of Moscow Transplantology ICU from December 2017 to January 2019. The strains were isolated from blood (n = 17), wounds (n = 14), skin (n = 15), respiratory system (n = 9), and from the surfaces of the areas closest to five patients (n = 8) (Table 1). K. pneumoniae strains were not collected from the environment of remaining 25 patients. Repeated examinations were performed for the patients with more severe infections. For example, strains of the patient B were collected on January 9, 2018, from the wound, blood, and environment; on January 10, 2019, from pharynx; and on January 12, 2019, from pleura. It was shown that these strains belonged to ST395 (wound, blood), ST39 (pharynx, environment), and ST3346 (pleura).
Phenotypes of ABR
Based on the analysis of susceptibility to ABs, 55 (87.3%) strains were classified to XDR category, and the remaining 8 (12.7%) strains were classified to MDR category. Data on bacterial strains resistance to eight functional groups of ABs are given below. Four strains (6.3%) were resistant to 8 AB functional groups, 43 strains (68.3%) to 7 AB functional groups, 10 strains (15.9%) to 6 AB functional groups, and 6 strains (9.5%) to 5 AB functional groups. All 63 (100%) strains were resistant to penicillins, cephalosporins, and fluoroquinolones, 62 strains (98.4%) to aminoglycosides, 53 strains (84.1%) to carbapenems, 52 strains (82.5%) to sulfonamides, 49 strains (77.8%) to tetracyclines, and 18 strains (28.6%) to polymyxins. Noteworthy is the fact that most of the strains were susceptible to three ABs: 33 strains (52.4%) to colistin, 41 strains (65.1%) to amikacin, and 44 strains (69.8%) to tigecycline (Table 2).
Genetic Lines, Capsular Types, Antibacterial Resistance Genes, and Antibacterial Resistance Phenotypes of K. pneumoniae Strains
ND, K-type is not typable.
In bold are the functional groups taken into account to determine the MDR and XDR categories accordingly to German et al. (2018).
First case of detection in Russia.
Novel sequence type.
ST, sequence type; blaTEM, blaSHV, blaCTX-M, blaOXA, blaNDM, beta-lactamase genes; int1, class 1 integron integrase; PEN, penicillins; CEF, cephalosporins; CAR, carbapenems; AMI, aminoglycosides; QNL, fluoroquinolones; SUL, sulfonamides; TET, tetracyclines; POL, polymyxins.
Genetic determinants of ABR and virulence
It was shown that K. pneumoniae strains carried beta-lactamase genes of blaSHV-type (63 strains, 100%), blaCTX-M-type (61 strains, 96.8%), blaTEM-type (54 strains, 85.7%), and carbapenemase genes blaOXA-type (52 strains, 82.5%) and blaNDM-type (2 strains, 3.2%). Carbapenemase genes of blaKPC-, blaVIM-, and blaIMP-types were not detected. Both ESBL and non-ESBL beta-lactamase genes were identified in the study: the ESBL genes blaSHV-2a, blaSHV-27, blaSHV-67, blaCTX-M-14, and blaCTX-M-15; and the non-ESBL genes blaSHV-1, blaSHV-11, blaSHV-187, and blaTEM-1; as well as carbapenemase genes were identified: blaOXA-48 and blaNDM-1. The set of four beta-lactamase gene types blaSHV+blaCTX-M+blaTEM+blaOXA was identified in 43 strains (68.3%). Four variants of three beta-lactamase gene types were detected in 20 strains (31.7%): blaSHV+blaCTX-M+blaTEM in 9 strains (14.3%), blaSHV+blaCTX-M+blaOXA in 7 strains (11.1%), blaSHV+blaTEM+blaOXA in 2 strains (3.2%), and blaSHV+blaCTX-M+blaNDM in 2 strains (3.2%) (Table 2).
Moreover, class 1 integrons were identified in 19 strains (30.2%), wherein the 4 strains (6.3%) carried two integrons simultaneously. Five types of the class 1 integron insertions carrying gene cassettes providing resistance to aminoglycosides and sulfonamides were identified: aacA4 (n = 2), dfrA1-orfC (n = 6), aadB-aadA1 (n = 9), dfrA15-aadA1 (n = 3), and dfrA12-orfF-aadA2 (n = 1). Class 2 integrons were not detected.
It was shown that major strains (n = 61) had the same set of four K. pneumoniae virulence genes: wabG, fimH, uge, and allS. Two strains carried additionally kfu gene. The rmpA, aer and allR genes were not detected.
Genetic lines of K. pneumoniae
Six known K. pneumoniae STs, ST395 (n = 44), ST377 (n = 3), ST307 (n = 4), ST13 (n = 2), ST39 (n = 2), and ST3346 (n = 1), and novel sequence-type ST3551 (n = 7) were identified in the study. Interestingly, more than one STs were isolated from three patients: ST395 and ST13 from the patient A; ST395, ST39, and ST3346 from the patient B; and ST395 and ST377 from the patient K. Only one ST of K. pneumoniae was isolated from the remaining patients. A total of eight K. pneumoniae strains were recovered from the environmental samples associated with five patients, including ST395 (n = 6), ST377 (n = 1), and ST39 (n = 1). In each case, environmental isolates belonged to the same ST as isolates collected from the associated patient (Table 1).
Most K. pneumoniae strains of ST395 (n = 41) displayed XDR phenotype. All strains were resistant to five to eight functional classes of ABs; however, two thirds of the strains were susceptible to amikacin, tigecycline, or colistin. The strains carried four combinations of beta-lactamase genes, most of them (n = 25) were classified as capsular type K2, and the rest strains to capsular types K47, KL39, and KL102/149/155.
K. pneumoniae strains of ST377 were classified to XDR phenotype; they were resistant to five to six functional classes of ABs. Among them, blaOXA-48-negative strains were susceptible to tetracyclines, polymyxins, aminoglycosides, and imipenem, but blaOXA-48-positive strain was susceptible only to tetracyclines and polymyxins. The strains carried two combinations of beta-lactamase genes: blaSHV-27+blaTEM-1+blaCTX-M-15 and blaSHV-1+blaTEM-1+blaCTX-M-15+blaOXA-48 associated with K2 and KL102/149/155 types, respectively.
K. pneumoniae ST307 strains demonstrate the XDR phenotype, resistance to five to seven functional classes of ABs. All strains were susceptible to polymyxins, and two strains additionally to carbapenems. The strains carried two combinations of beta-lactamase genes: blaSHV-1+blaTEM-1+blaCTX-M-15 and blaSHV-1+blaTEM-1+blaCTX-M-15+blaOXA-48. All strains of ST307 were classified as capsular type KL102/149/155.
MDR phenotype was identified for both K. pneumoniae ST13 strains, which were resistant to six functional classes of ABs, including reserve drug colistin. They were susceptible to meropenem and amikacin. These strains carried beta-lactamase genes blaSHV-187+blaTEM-1+blaCTX-M-15+blaOXA-48; they were attributed to capsular type K3.
Two K. pneumoniae ST39 strains displayed MDR phenotype, they were resistant to six ABs functional classes including colistin, but susceptible to meropenem and amikacin. The strains carried three beta-lactamase genes blaSHV-1+blaTEM-1+blaCTX-M-14 and belonged to capsular type K23.
One K. pneumoniae ST3346 strain with MDR phenotype was resistant to six ABs functional classes including colistin and susceptible to meropenem and amikacin. This strain carried four beta-lactamase genes blaSHV-2a+blaTEM-1+blaCTX-M-14+blaOXA-48 and belonged to capsule type K23 (Tables 1 and 2).
Novel K. pneumoniae sequence-type ST3551 was identified for seven strains isolated from five patients in July, August, November, and December of 2018. The ST3551 was characterized by a unique allelic profile gapA3, infB4, mdh6, pgi1, phoE7, proB4, and ton479 (BIGSdb ID 8130). This ST has six alleles that are identical to such of ST147, only the ton gene is different because of a point mutation G237A, compared with the allele ton38 of the ST147, thus ST3551 was attributed to the clonal group CG147. All K. pneumoniae ST3551 strains were classified as XDR pathogens, resistant to six to eight ABs functional classes. Two strains were not susceptible to any of used ABs, one strain was susceptible only to tigecycline, two strains to tigecycline and tetracycline, one strain to tigecycline, tetracycline, and colistin, and one strain to tigecycline, tetracycline, colistin, and polymyxin. These strains carried three beta-lactamase genes blaSHV-67+blaCTX-M-15+blaOXA-48, they were attributed to capsular type K14/64 (Table 2 and Fig. 1).
Antibacterial resistance prevalence among Klebsiella pneumoniae of sequence types identified at the study: PEN, penicillins; CEF, cephalosporins; CAR, carbapenems; AMI, aminoglycosides; QNL, fluoroquinolones; SUL, sulfonamides; TET, tetracyclines; POL, polymyxins.
Phylogenetic tree
The phylogenetic analysis of the MLST profiles of seven STs identified in this study showed the presence of two clusters. The first of them included the novel ST3551 and the reference sequence-type ST147. The second cluster includes six STs: ST13, ST39, ST307, ST377, and ST395, which were divided into four groups: the first included ST307, the second included ST13, the third included ST39 and ST3346, and the fourth included ST395 and ST377. It should be noted that most of the strains of this study (n = 47) were attributed to the fourth group. The STs of the third group, ST39 and ST3346, were different from each other on a point substitution T429C in mdh gene (mdh2 and mdh1, respectively) and belonged to the same clonal group CG39. STs of the fourth group, ST395 and ST377, were phylogenetically more distant from each other, and they carried nine point substitutions in the genes: gap (T213C, C285T), inf (C291T), pgi (T150C), pho (A54G), rpo (C87T, A130G, C333T), and ton (C370G). Thus, seven STs belonging to five K. pneumoniae phylogenetic groups were identified in the study (Fig. 2).
Phylogenetic tree of K. pneumoniae sequence types identified in this study. The asterisk indicates the reference sequence.
Discussion
The results of the study confirmed the negative trend of the recent decades—increasing prevalence of MDR strains among nosocomial K. pneumoniae. Most analyzed strains were classified as XDR; seven STs were identified. Seven XDR strains and one MDR strain were isolated from the nearest environment of the patients. Unfortunately, in the framework of this study, we cannot answer the question if the patients acquired their infections through the contact with the contaminated environment. The ICU patients were not examined on the carriage of K. pneumoniae before admission. The nearest environment of the patients included items of their clothing, bedding, and the surface of the bedside furniture. We have no data about bacterial contamination of room air, room conditions, and so on. Only five K. pneumoniae strains were isolated from the areas closest to the patients and attributed to the same genetic lines as the strains collected from the corresponding patient. K. pneumoniae strains were not collected from the areas closest to remaining 25 patients.
This study was the first research conducting MLST study of K. pneumoniae clinical strains in the Moscow Transplantology ICU. The prevalent K. pneumoniae genetic line was ST395, a global epidemically successful clone, spreading OXA-48-, KPC-, and IMP-type carbapenemase genes in Europe and Asia.25–27 Beta-lactamase genes blaKPC, blaVIM, and blaIMP were not detected in the strains of this study. Previously, the blaOXA-48 gene was identified in K. pneumoniae of ST395 in Russia.28,29 The first case of carbapenemase gene blaNDM-1 detection in K. pneumoniae ST395 in Russia is presented in this study, while previously the blaNDM-1 gene was detected in K. pneumoniae of ST340 and ST147.28,30
Other K. pneumoniae STs identified in this study are also listed among the most epidemically significant in the world. K. pneumoniae ST307 was recently described as a unique genetic line, which was considered to be a candidate clone of increased risk for carbapenemase transmission, due to new virulence factor associated with glycogen synthesis, which can provide pathogen a high degree of adaptation to both hospital environment and human body. 31 Previously, K. pneumoniae ST307 strain carrying two carbapenemase genes, blaNDM-1 and blaOXA-48, located on two plasmids of different incompatibility groups IncL/M and IncFII, was described in France. 32 K. pneumoniae strains of this ST carrying carbapenemase genes blaOXA-48 and blaKPC-2,-3 isolated in Russia were found in the BIGS database on the date July 19, 2019. In this study, we also isolated the K. pneumoniae ST307 strain carrying the blaOXA-48 carbapenemase gene.
The sequence-type ST13 was described since the 1980s in many regions of the world as the widely spread K. pneumoniae genetic line. In recent years, MDR strains of this genetic line carrying the TEM-3 and DHA-1 beta-lactamase genes were identified in Spain, and OXA-48-producing strains in Finland, Ireland, and Algeria.8,33–35 In this study, we determined the sequence-type ST13 for two MDR K. pneumoniae strains carrying blaCTX-M-15 and blaOXA-48 genes as the first case in Russia.
K. pneumoniae ST39 is globally distributed carrier of ESBL in Africa,36,37 of VIM-1 carbapenemase in Spain, 38 of KPC-2 carbapenemase in China and Finland,39,40 and of OXA-48 carbapenemase in Israel. 41 In our study, two K. pneumoniae strains of ST39 carrying the gene for epidemically significant ESBL CTX-M-14 were first identified in Russia.
K. pneumoniae strain of relatively new ST3346 belonging to CG39 was first isolated from the healthy person in Africa in 2017 (BIGSdb ID 7237). In our study, we describe the first case of K. pneumoniae ST3346 collected in Russia in 2018; the carbapenemase gene blaOXA-48 and the ESBL gene blaCTX-M-14 in K. pneumoniae of ST3346 are presented for the first time.
Sequence-type ST377 is known as a rarely occurring K. pneumoniae clone bearing carbapenemase genes. The BIGS database contains information about the ST377 isolates obtained from France, and ST377 was also previously described in Russia. 42 In the present study, one strain of K. pneumoniae ST377 carrying the blaOXA-48 carbapenemase gene was characterized.
Novel K. pneumoniae sequence-type ST3551 was attributed to the clonal group CG147, the founder of which ST147 is one of the main international high-risk nosocomial clones, due to its ability to survive on environmental surfaces, to form biofilms and displaying resistance to disinfectants and antiseptics. 43 This clone is known to be associated with carbapenemase VIM-1, NDM-1, and KPC-2 genes in many countries. 44 Recently, a unique K. pneumoniae strain of ST147 was reported, possessing not only an XDR phenotype, but also hypermucoid and hypervirulence. 45 The strains of ST3551 isolated in this study carried blaSHV-67 and blaCTX-M-15 ESBL genes and blaOXA-48 carbapenemase gene, as well as class 1 integron.
Thus, the genetic mechanism of XDR phenotype of the major strains in our study was associated with blaSHV-, blaCTX-M-, blaTEM-, blaOXA-, and blaNDM-type beta-lactamase genes, and class 1 integrons; this conclusion coincides with the opinion of other authors. 46 Noteworthy, five ESBL genes (blaCTX-M-14, blaCTX-M-15, blaSHV-2a, blaSHV-27, and blaSHV-67) and two carbapenemase genes (blaOXA-48 and blaNDM-1) were identified.
Category of CR K. pneumoniae attracts the special attention of researchers all over the world. The proportion of CR strains in our study was more than 80%, which is comparable with the data (74%) published in Italy. 47 Of particular interest are carbapenemase-positive strains of genetic lines ST395OXA-48, ST377 OXA-48, ST307 OXA-48, novel ST3551OXA-48, and three genotypes that were not described previously in Russia: ST13OXA-48, ST3346OXA-48, and ST395NDM-1.
An additional characteristic reflecting the genetic diversity of the strains under study is their belonging to seven capsular types: K2, K3, K14/64, K23, KL39, K47, and KL102/149/155. Interestingly, among the strains of the same ST, different K-types were detected: ST395 was associated with K2, KL39, K47, and K102/149/155 and ST377 was associated with K2 and K102/149/155. At the same time, among the strains of the same capsular type, different STs were detected: K2 was associated with ST395 and ST377; K102/149/155 with ST395, ST377, and ST307; K23 with ST39 and ST3346. This observation reflects the fact that the genes of capsule cluster do not belong to the allelic profile of MLST typing. It was noted that one of the mechanisms for diversity of a capsular cluster is genetic recombination within this cluster. 48 Phylogenetic analysis confirmed the extremely high level of genetic diversity of K. pneumoniae MDR strains isolated for about 1 year in a high-tech Transplantology ICU. Seven STs formed five phylogenetic groups. A novel K. pneumoniae sequence-type ST3551 was assigned to a genetically distant group from other STs in this study.
The genetic lines of K. pneumoniae identified in this study (CG258, CG307, CG39, CG13, and CG147) were described as the international high-risk clones. It was reported from other studies that such bacteria have ability to persist and disseminate in both hospital environment and human body due to increased adaptive properties, biofilm formation, and displaying resistance to disinfectants and antiseptics.25–27,31,33–41,43,44 Apparently, the immunity suppression and preliminary multiple antibiotic therapy were risk factors for colonization of the Transplantology ICU patients by MDR K. pneumoniae strains.49,50
Conclusion
The study characterized the clinical strains of K. pneumoniae isolated from the Transplantology ICU, among which the majority strains were XDR and the rest were MDR. The genetic mechanism of MDR of these strains is the carriage of beta-lactamase genes including carbapenemase genes, and class 1 integrons. We identified the epidemically significant international STs of K. pneumoniae ST13, ST39, ST307, ST395, and ST3346. The genetic lines ST395NDM-1, ST13OXA-48, ST3346OXA-48, ST39CTX-M-14, and novel sequence-type ST3551OXA-48 were first described in Russia.
Hospital pathogen genotyping is important for clinical epidemiologists to control and prevent infections associated with medical care. Identification of the novel for Russia clonal groups of CR- and ESBL-producing K. pneumoniae confirms ongoing genetic processes of the MDR and XDR spread among gram-negative bacteria in high-tech ICUs.
Footnotes
Acknowledgment
We thank the team of curators of the Institute Pasteur MLST and whole-genome MLST databases for curating the data and making them publicly available on the website.
Disclosure Statement
No competing financial interests exist.
Funding Information
The study was financially supported by the State Research Center for Applied Microbiology and Biotechnology. The work of N.K.F. and N.V.V. consisting of genotyping Klebsiella pneumoniae was also supported by the Russian Science Foundation (Grant No. 15-15-00058).