RU2797025C1 - Klebsiella pneumoniae bacterial strain used as a test culture for the detection of the ndm gene by polymerase chain reaction for accurate diagnosis and prescription of antibacterial therapy for klebsiella infections - Google Patents

Abstract

FIELD: microbiology; biotechnology.

SUBSTANCE: Klebsiella pneumoniae 1970_kpn bacterial strain producing New Delhi metal-β-lactamase and possessing multiple resistance to antibiotics deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures (GKPM-Obolensk) under registration number B-9981.

EFFECT: invention provides expansion of the arsenal of reference strains when monitoring the spread of NDM-type carbapenemase producers and can be used in detecting Klebsiella infections to adjust therapy.

1 cl, 5 tbl, 2 ex

Description

SUBSTANCE: invention relates to medical microbiology and concerns the use of Klebsiella pneumoniae 1970_kpn culture as a reference strain in PCR diagnostics for NDM-like metallo-p-lactamase genes.

The niches of K. pneumoniae are divided into the environment, mammalian mucosal surfaces, medical equipment, and personnel. It is present in small amounts as a saprophyte in humans, as well as in the normal flora of the oral cavity, skin and intestines. This pathogen occupies a leading role in the etiology of a wide range of community-acquired and hospital-acquired human infections, which poses a serious threat to public health. Outbreaks of classic Klebsiella are usually associated with health care, prolonged hospitalization, or antibiotic therapy. K. pneumoniae causes a variety of infections including pneumonia, gastrointestinal lesions, eye infections, sepsis, urinary tract infections, bacteremia, meningitis, liver abscesses, skin infections, primary liver abscesses, and affects middle-aged and older immunocompromised patients.

The biggest problem with classic K. pneumoniae is resistance to carbapenems. In 2013, the Centers for Disease Control and Prevention published a list of priority pathogens that pose an immediate threat to public health, including Enterobacteriaceae resistant to carbapenems, which is today the “last line of defense” in the treatment of the most severe infections [1]. In 2019, they continued to focus on this [1, 2]. In parallel, in 2014, WHO announced that carbapenem-resistant K. pneumoniae had emerged in all its regions, with some of them exceeding 50% prevalence [4]. In 2017, WHO also confirmed the spreading threat of carbapenem-resistant Enterobacteriaceae, as well as some other pathogens, with a critically high priority for the development of new antibiotics [5].

NDM belongs to Amber class B metallo-β-lactamases. The first variant was first described in Sweden in 2009 in a patient who traveled to India where he acquired a urinary tract infection caused by carbapenem-resistant Klebsiella pneumoniae. Since then, NDM producers have begun to emerge in most regions of the world. Since then, more than 40 NDM-type carbapenemase variants have been identified. Metallo-β-lactamases are insensitive to clinically available inhibitors, which limits therapeutic options. The mechanism of antibiotic resistance is that two zinc ions present in the active site of metallo-β-lactamases cause hydrolysis of the amide bond in the β-lactam ring, inactivating the antibiotic.

The applicant has not identified sources containing information on technical solutions identical to the present invention, which allows us to conclude that it meets the criterion of "novelty".

The applicant is not aware of any publications that would contain information about the impact of the distinctive features of the invention on the achieved technical result. In this regard, according to the applicant, it can be concluded that the proposed technical solution meets the criterion of "inventive step".

However, there is a work in which the authors found the metallo-β-lactamase NDM-29 gene in E. coli isolated from the bile of a patient in 2019 in China. It turned out that the region carrying blaNDM-29 is located on the transposon, which indicates the risk of the spread of resistance caused by NDM-29. Transformation experiments showed no difference in antibiotic susceptibility of the strain compared to a transformant containing a plasmid with a localized NDM-1 gene variant. It was also confirmed that the presence of the NDM-29 gene does not increase the biological cost of bacterial resistance [7]. However, in this New Delhi study, metallo-P-lactamase was found in a strain of Escherichia coli, which does not allow us to characterize the mechanisms of distribution of this type of carbapenemase among the pathogens of Klebsiele infections.

In another paper, the authors described "high risk" clones carrying hybrid resistance/virulence plasmids found in the UK. In particular, they identified twelve isolates belonging to different clonal lines, carrying carbapenemase genes and showing resistance to almost all antibiotics tested. The virulence plasmids of three members of ST383 carried bla _NDM-5 and seventeen other resistance genes. A common area of antibiotic resistance is noted in the virulence/resistance plasmids of ST 383 and ST48 isolates, suggesting transposable element-mediated recombination [6]. However, in this work, the copy number of the NDM gene was not assessed, which is necessary for PCR sensitivity analysis.

In a study by a research team from Italy, the authors collected the genomes of 117 NDM-producing extensively drug-resistant strains of K. pneumoniae over a period of 20 months from 76 patients at several medical facilities in southeastern Tuscany. All isolates belonged to the high-risk ST-147 clone and were generally insensitive to all first-line antibiotics. In this study, there were signs of selective pressure based on genes associated with drug resistance. The authors noted the emergence of variants associated with resistance to either tigecycline or colistin, which are almost the only effective treatments other than combinations of antibiotics [3]. The limitation of this work is the lack of data on the copy number of the New Delhi metallo-β-lactamase gene, which is necessary for the analysis of the analytical sensitivity of PCR diagnostics.

The objective of the invention is to obtain a test culture used in the detection of the NDM gene by polymerase chain reaction.

The technical result of the present invention is to obtain a strain of Klebsiella pneumoniae producing New Delhi metallo-β-lactamase and characterized by multiple resistance to antibiotics.

This result is achieved by isolating the Klebsiella pneumoniae 1970_kpg strain carrying a resistance plasmid with a localized, newly discovered NDM-29 gene variant.

The essence of the invention is as follows.

The Klebsiella pneumoniae 1970-kpn strain was isolated in April 2018 from a patient's urine sample at the National Medical Research Center of Oncology named after. N.N. Petrov, St. Petersburg. The strain was deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures (GKPM-Obolensk) under registration number B-9981.

Generic name Klebsiella - small gram-negative coccobacilli, 1-2 μm × 0.5-0.8 μm in size do not form spores, are immobile, capable of forming a capsule, arranged singly, in pairs and in clusters, easily stained with aniline dyes. Chemoorganoheterotroph, facultative anaerobe. Oxidase negative, catalase positive. Under the action of antibiotics, they can form L-forms.

The genus Klebsiella currently includes 14 species, including Klebsiella pneumoniae (Schroeter, 1886).

Cultivated on simple nutrient media at 37°C. On dense nutrient media, it forms shiny, convex, slimy colonies; on liquid media, it causes diffuse turbidity, a film, and a parietal ring. On Endo and Ploskirev media, they form red colonies with a metallic sheen (lactose positive). On blood agar, K. pneumoniae colonies do not show hemolysis. Temperature range 12-43°C, optimum pH 7.2.

It occurs naturally in the soil, about 30% of strains can fix nitrogen under anaerobic conditions. K. pneumoniae is able to metabolize glycerol as its sole carbon source. They ferment sugars such as glucose, lactose, sucrose, mannitol to produce acid and gas. K. pneumoniae are urease positive, indole negative, methyl red negative, citrate positive, and Voges-Proskauer positive.

There are two types of antigenic structures: the capsular (K) antigen, subdivided into 80 serotypes, and the lipopolysaccharide somatic (O) antigen, subdivided into 8 different serotypes. K. pneumoniae 1970_kpn strain to KL20, 02vl. Virulence in a septic mouse model LD ₅₀ =10 ⁴ . The copy number of the NDM gene was 202 copies per µl.

For taxonomic identification of the strain, MALDI-TOF mass spectrometry was used, as well as whole genome sequencing using a hybrid approach, including sequencing on the Illumina MiSeq and Oxford Nanopore platforms.

Genetic characteristics:

The chromosome contains the following virulence genes: entABES, fepBCDG, ecpABCDER, fimABCEH, kdsA, silA. The strain also contains 3 plasmids of different types of incompatibility. Plasmid IncFIB/IncHIl B carries the following resistance genes: sul2, armA, msr(E), mph(E), qnrSl, aph(3')-VI, blaNDM-29, dfrA5, sull; and virulence iutA, iucABCD, prmpA, prmpA2, pegl344, cobW, luxR pagO, ydjA, shiF. Plasmid IncFIB carries the trimethoprim resistance gene (dfrA5). Plasmid IncR carries the following resistance genes blaTEM-1A, blaOXA-9, aadA, aac(6')-Ib.

Storage conditions: freeze-dried culture at a temperature of 4±2°C in a place protected from direct sunlight at a relative humidity of not more than 60% for 2 years. After storage, the culture forms uniform colonies.

Example 1

Experiments were carried out on the detection of New Delhi metallo-β-lactamase by real-time polymerase chain reaction (PCR) in 563 clinical strains of K. pneumoniae isolated in 2019-2022 from intensive care patients and demonstrating multiple resistance to antibiotics.

Genomic bacterial DNA was isolated using the DNA-Sorb-B kit (AmpliSens, Russia) according to the manufacturer's protocol. Real-time PCR for the detection of carbapenemase genes was performed using a CFX96 Touch device (Bio-Rad, USA). Primers and probes for genes encoding NDM type carbapenemase with the length of amplification products and their melting points are presented in Table 1. Primers were synthesized in Evrogen (Russia), probes - in Sintol (Russia). The NDM genes are registered by the Cy5 fluorophore channel (red).

The reaction was carried out using Dream Taq Hot Start PCR Master Mix according to the manufacturer's recommendations. Temperature, in accordance with the annealing temperature of primers and probes, and component protocol are presented in tables 2 and 3, respectively.

The strain K. pneumoniae 1970_krp under the registration number GKPM-Obolensk B-9981 was used as a reference strain and gave a characteristic amplification curve, thereby confirming the correct operation of the detection mixture and the master mix. It was experimentally established that NDM producers accounted for 5.5% (n=31) of the total sample.

Example 2

Experiments were carried out on the detection of New Delhi metallo-β-lactamase by real-time multiplex PCR using the commercial kit Amply Sens in 117 clinical strains of K. pneumoniae, isolated from 19 hospital-type medical institutions during 2017 from a variety of clinical material, received from intensive care patients.

Bacterial DNA was isolated using the DNA-sorb B kit (Interlabservis, Russia) in accordance with the manufacturer's instructions. The genes for clinically significant metallo-β-lactamases were detected by real-time PCR using the AmplySens®MDR MBL-FL kit (bla _VIM , bl _IMP , bla _NDM ).

The process began with the preparation of a mixture of PCR mix-2-FRT (300 μl) with TaqF polymerase (30 μl). The contents were remixed with the addition of PCR mix-1-FRT (1200 μl). The calculation was carried out in accordance with the ratio of DNA to PCR mixture for 1 reaction: 15 μl of PCR mixture to 10 μl of DNA. Further, the reagents were added in accordance with Table 4.

The detection of the fluorescent signal was carried out using the Real-Time PCR Detection System with the programmed temperature protocol indicated in Table 5.

Detection was carried out through four channels:

- through the channel for the FAM fluorophore, a signal is recorded indicating the accumulation of the amplification product of the MBL gene fragments of the VIM group;

- through the channel for the JOE fluorophore, a signal is recorded indicating the accumulation of the product of amplification of fragments of MBL genes of the IMP group;

- through the channel for the ROX fluorophore, a signal was recorded indicating the accumulation of the internal control DNA amplification product;

- through the channel for the Cy5 fluorophore, a signal was recorded indicating the accumulation of the product of amplification of fragments of the MBL genes of the NDM group.

Graphical display of PCR results was performed using CFX96 Maestro Analysis Software.

The strain K. pneumoniae 1970_kpn under the registration number GKPM-Obolensk V-9981 was used as a reference strain and gave a characteristic amplification curve, thereby confirming the correct operation of the detection mixture. It was experimentally established that NDM producers accounted for 43.6% (n=51) of the total sample.

Can be used to select antibacterial agents suitable for effective control of nosocomial strains of K. pneumoniae. EFFECT: invention makes it possible to increase efficiency in combating nosocomial Klebsiella infections.

It can be used as a test culture to monitor the distribution of NDM-type carbapenemase producers in the Russian Federation in Klebsiella isolated in hospital and out-of-hospital environments.

Literature

1. ANTIBIOTIC RESISTANCE THREATS in the United States [Web Page]. - CDC, 2013. - Available online: https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf.

2. CDC. ANTIBIOTIC RESISTANCE THREATS IN THE UNITED STATES / CDC // -2019. - -.

3. Martin, M. J.; Corey, B. W.; Sannio, F.; Hall, L.R.; MacDonald, U.; Jones, B.T.; Mills, E.G.; Harless, C; Stam, J.; Maybank, R.; et al. Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy / Martin, M.J.; Corey, B. W.; Sannio, F.; Hall, L.R.; MacDonald, U.; Jones, B.T.; Mills, E.G.; Harless, C; Stam, J.; Maybank, R.; et al. // Proc Natl Acad Sci USA. - 2021. - 118. -.

4 Organization, W.H. ANTIMICROBIAL RESISTANCE Global Report on Surveillance / Organization, W.H. // - 2014.- -.

5 Organization, W.H. Critically high priority for developing new antibiotics / Organization, W.H. // - 2017. - -.

6 Turton, J.; Davies, F.; Turton, J.; Perry, C; Payne, Z.; Pike, R. Hybrid Resistance and Virulence Plasmids in "High-Risk" Clones of Klebsiella pneumoniae, Including Those Carrying blaNDM-5 / Turton, J.; Davies, F.; Turton, J.; Perry, C; Payne, Z.; Pike, R. // Microorganisms. -2019. - 7. -.

7. Zhu, Y.; Jia, X.; Jia, P.; Li, X.; Yang, Q. Genetic and Phenotypic Characterization of the Novel Metallo-beta-Lactamase NDM-29 From Escherichia coli / Zhu, Y.; Jia, X.; Jia, P.; Li, X.; Yang, Q. // Front Microbiol. - 2021. - 12. - 743981.

Claims (1)

The Klebsiella pneumoniae 1970_kpn strain, which is used as a reference strain in PCR diagnostics for NDM-like metallo-β-lactamase genes to adjust therapy in case of detection of Klebsiella infections, deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures (GKPM-Obolensk) under registration number B- 9981.