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The Genome Structure of Ciprofloxacin-Resistant Mycoplasma Hominis Clinical Isolates
E. A. Kolesnikova*, N. F. Brusnigina, M. A. Makhova, A. E. Alekseeva
Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology, Federal Service for Surveillance on Customers Rights Protection and Human Wellbeing, Nizhniy Novgorod, 603950 Russia *E-mail: shmelevael@yandex.ru
Received October 4, 2019; in final form, February 19, 2020 DOI: 10.32607/actanaturae.10941
Copyright © 2020 National Research University Higher School of Economics. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT The genome structure of three ciprofloxacin-resistant Mycoplasma hominis clinical isolates was studied using next-generation sequencing on the Illumina platform. The protein sequences of the studied Mycoplasma strains were found to have a high degree of homology. Mycoplasma hominis (M45, M57, MH1866) was shown to have limited biosynthetic capabilities, associated with the predominance of the genes encoding the proteins involved in catabolic processes. Multiple single-nucleotide substitutions causing intraspecific polymorphism of Mycoplasma hominis were found. The genes encoding the efflux systems - ABC transporters (the ATP-binding cassette superfamily) and proteins of the MATE (multidrug and toxic compound extrusion) family - were identified. The molecular mechanism of ciprofloxacin resistance of the Mycoplasma hominis M45 and M57 isolates was found to be associated with the Ser83Leu substitution in DNA gyrase subunit A. In the Mycoplasma hominis MH1866 isolate it was related to the Lys144Arg substitution in topoisomerase IV subunit A. KEYWORDS Mycoplasma hominis, genome structure, antibiotic resistance mechanisms, gyrA and parC genes, ABC transporters, MATE.
INTRODUCTION
Mycoplasma hominis is one of the most common members of the class Mollicutes. Its characteristic features include the absence of a rigid cell wall; an ability to persist on the eukaryotic cell membrane; the small size of their genome; genetic and cell polymorphism; limited metabolic pathways; and antimicrobial resistance (AMR) to drugs that aim to inhibit cell wall biosynthesis [1].
The Mycoplasma hominis strains are known to predominantly colonize the urogenital tract in men and women, both healthy ones and those suffering from inflammation (urethritis, cervicitis, vaginitis, bacterial vaginosis, etc.). The ability of Mycoplas-ma hominis to colonize the upper respiratory tract and cause respiratory infections in infants has been proved [1, 2].
Recent data in Russian and foreign publications demonstrate the prevalence of urogenital mycoplas-mas resistant to fluoroquinolones and macrolides, the most commonly prescribed antibiotics in the treatment of inflammatory diseases of the pelvic organs [3-5]. Monitoring antimicrobial resistance in bacteria (including Mycoplasma hominis), the pathogens that
cause reproductively relevant infections, is a pressing biomedical problem. To resolve it, one needs to study the fundamentals of their pathogenicity, resistance, and adaptation to stressful environments. Modern molecular genetic technologies, next-generation sequencing (NGS) in particular, have made it possible to get closer to understanding these processes. A French research team led by S. Pereyre was the first to sequence and decode the complete nucleotide genome sequence of Mycoplasma hominis (Mycoplasma hominis ATCC 23114, GenBank accession number FP236530.1) in 2009 [6]. Today, the international GenBank database contains information on complete genome sequences for 23 Mycoplasma hominis strains. It should be noted that studying the evolutionary diversity of the Mycoplasma hominis population both in Russia and abroad is a challenge, because there are no data on the peculiarities of their genome structure, including pathogenic factors and resistance among the M. hominis strains.
The aim of this work was to analyze the genome structure of the ciprofloxacin-resistant M. hominis clinical isolates found in women with inflammatory diseases of the urogenital tract.
EXPERIMENTAL
Our study subjects were three M. hominis clinical isolates (M45, M57, and MH1866) found in epithelium scraped from the cervical canal of women suffering from inflammatory diseases of the urogenital tract. The women had provided a written informed consent to participate in the study. Commercial differential diagnostic liquid environments manufactured by the Central Scientific Research Institute of Epidemiology, Scientific Research Institute of Epidemiology and Microbiology, Federal Service for Monitoring of Customers Rights Protection and Human Wellbeing (registration number FSR 2008/03366), were used to detect and identify the mycoplasmas, as well as to determine their antibiotic susceptibility pattern. All the studied strains were ciprofloxacin-resistant. The results of a multi-year microbiological monitoring of the prevalence and antibiotic resistance of urogenital mycoplasmas isolated from women and men (both healthy and suffering from inflammatory diseases of the urogenital tract) were reported previously [7-10]. DNA isolation and purification was performed using an AmpliPrime DNA-sorb-V kit (Central Scientific Research Institute of Epidemiology, Federal Service for Monitoring of Customers Rights Protection and Human Wellbeing, Moscow, Russia). Whole genome sequencing was performed on a MiSeq sequencer (Illumina, USA). DNA concentration in the samples was estimated using a Qubit fluorimeter (Invitrogen, Austria). The DNA library for sequencing was prepared using a Nextera XT kit (Illumina, USA). Sequencing was performed using a MiSeq Reagent Kit v2 (Illumina, USA) for 500 cycles. The reference was the whole genome sequence of the Mycoplasma hominis ATCC 23114 strain (GenBank accession number FP236530.1). The nucleotide sequences were aligned using the embedded software of the MiSeq sequencer (Isis version 2.6.2.3). Visualiza-
tion and analysis of the acquired data were performed using the UGENE Unipro [11] and MEGA 7.0 software [12]. The genome annotation was carried out with the help of Rapid Annotation using the Subsystem Technology (RAST) server [13] and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (https://www. ncbi.nlm.nih.gov/genome/annotation_prok/). Phy-logenetic analysis of the whole-genome nucleotide sequences for the studied strains was conducted using the REALPHY web service [14] Online tool, version 1.12 (https://realphy.unibas.ch/fcgi/realphy). The analysis included all the genome nucleotide sequences of Mycoplasma hominis deposited in the RefSeq NCBI database (https://www.ncbi.nlm.nih.gov/refseq). Phy-logenetic trees were built with the neighbor-joining algorithm [15] in the MEGA7.0 software [12].
RESULTS
The whole-genome nucleotide sequences of M. hom-inis have been deposited in the international database NCBI GenBank under the accession numbers MRAY00000000 (M. hominis M45), MRAX00000000 (M. hominis M57), and QOKOOOOOOOOO (M. hominis MH1866). The source archives of the reads are available under the numbers SUB 6713744 (M. hominis M57), SUB 6713764 (M. hominis M45), and SUB 6713769 (M. hominis MH1866).
Sequencing and assembly of the initial reads made it possible to collect between 18 (MH1866 strain) and 27 (M45 and M57 strains) contigs. It is most likely that the gaps encountered during genome mapping of the M. hominis isolates were associated with absence of this region in the original archive of the reads. The size of the genome of the studied strains varied from 633,286 base pairs (M57) to 642,227 base pairs (M45); the GC content was 27.2%. The key metrics of the genome assembly of M. hominis are shown in Table.
Structural analysis of the genome of M. hominis clinical isolates (M45, M57, and MH1866)
Characteristic M. hominis isolates / GenBank accession number
MH45/ MRAY00000000 MH57/ MRAX00000000 MH1866/ Q0K000000000 Reference strain ATCC 23114
Assembly length, base pairs 642,227 633,286 639,787 665,445
Number of contigs 27 27 18 1
Coverage 599, 4945 599, 4945 599, 4945 -
Number of reads, million 3.8 3.8 3.8 -
N50 33,392 49,675 57,877 665,445
L50 6 4 4 1
% GC 27.2 27.2 27.2 27.1
Number of genes/pseudogenes 592/28 589/30 581/16 598/12
Number of coding sequences 546 543 546 557
Number of 16S-23S-5S operons 2 2 2 2
Precent protein sequence identity
Bidirectional best hit 100 99.9 99.8 99.5 99 98 95 90 80 70 60 50 40 30 20 10
Unidirectional best hit 100 99.9 99.8 99.5 99 98 95 90 80 70 60 50 40 30 20 10
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percent identity 6666666.220762
percent identity 2098.29
percent identity 6666666.201005
2098.27 6666666.220762 6666666.201005 2098.29
Contig Gene Lenghl Hit Contig все T Gene Hit Contig Gene Hit Contig |Bce * Gene
1 660 bi 1 1 bi I
2 110 bi 1 l bi 1 2 bi 2
3 49 bi 1 2 bi 1 3 bi 3
4 449 bi 1 3 bi l 4 bi 4
5 366 bi 1 4 bi 1 5 bi 5
6 71 bi 1 5 bi 1 6 bi 6
7 66 bi 1 6 bi 1 7 bi 7
8 258 bi 1 7 bi 1 8 bi 8
9 256 bi 1 8 bi 1 9 bi 9
10 229 bi 1 9 bi 1 10 bi 10
128 358 bi 4 74 bi 15 502 bi_ 11
129 818 bi 4 75 bi 15 503 bi II
130 38 bi 4 76 bi 15 504 bi_ 13
295 121 bi 13 218 bi 16 505 bi 197
296 603 bi 13 219 bi 16 506 bi 198
297 154 bi 13 220 bi 16 507 bi 199
329 185 bi 13 221 bi 16 508 bi 200
330 717 bi 13 222 bi 16 509 bi 201
331 95 bi 13 223 bi 16 510 |bS 202
332 285 bi 13 224 bi 16 511 bi 203
333 102 bi 13 225 bi 16 512 bi 204
334 194 bi 13 226 bi 16 513 bi 205
335 180 bi 13 227 bi 16 514 bi 206
336 528 bi 13 228 bi 16 515 bi 207
337 294 bi 13 229 bi 16 516 bi_ 208
338 496 bi 13 230 bi 16 517 Ibj_ 209
339 126 bi 13 231 bi 16 518 bi 210
340 72 bi 13 232 bi 16 519 bi 211
341 725 uni 13 233 bi 16 520 [bi 299
342 177 uni 13 232 bi 16 521 bi 300
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Fig. 1. Comparative analysis of the identity of the protein sequences of the M. hominis M45, M57, and MH1866 strains. The results were acquired using the RAST server
An analysis the data acquired using the PGAP (Prokaryotic Genome Annotation Pipeline) server showed that the general number of identified open reading frames was 543 for the M. hominis M57 strain and 546 for the M. hominis M45 and MH1866 strains, out of which 511 (94.1%), 531 (97.2%), and 534 (97.8%) are accordingly annotated as protein-coding genes. Sixteen pseudogenes were found in the genome structure of the M. hominis MH1866 strain; 30 pseudogenes, in the M. hominis M57 strain; and 28, in the M. hominis M45 strain. Most of the pseudogenes are incomplete nucleotide remnants with unknown functions. However, some of the M. hominis pseudogenes were found to contain premature stop codons (three for the M. hom-inis M57 strain) and reading frame shift mutations (four for the M. hominis MH1866 strain; two for the M. hominis M45 strain; and two for the M. hominis M57 strain). Two copies of the 16S-23S-5S rRNA operon were found in the genomes of all strains.
The genomes of the studied M. hominis strains were found to be highly homologous: the degree of protein sequence homology was about 95% (Fig. 1).
Since the genomes of all three strains have a similar structure, the diagram illustrating gene distribution
according to the functions of their product for the M. hominis MH1866 isolate is shown in Fig. 2.
The functions of the overwhelming majority of the genes in the M. hominis genome are related to the synthesis of protein (39.1%), DNA (13%), and RNA (11.6%) (Fig. 2). The system of central carbohydrate metabolism of the studied strains (the gene portion is 6.9%) is truncated and consists of individual components involved in the metabolism of pyruvate, pentose phosphates (precursors of ribose and deoxyribose), glucose, and lactose. It was found that the mycoplasma genome contains the pdP, deoD, deoB, and deoC genes encoding catabolic enzymes: pyrimidine nucleoside phosphoryl-ase, purine nucleoside phosphorylase, phosphopento-mutase, and deoxyriboaldolase, respectively. These enzymes participate in the catabolism of deoxyribose and deoxyribonucleoside. The M. hominis strains use the 2-deoxy-D-ribose portion of the 2'-deoxyribonucleo-sides resulting from a cascade of biochemical reactions during deoxyribose fermentation as the only source of carbon and energy. The presence of genes encoding enzymes of the purine nucleotide cycle (arginine deaminase (ArcA), ornithine transcarbamylase (ArgF), and carbamate kinase (ArqC)) allows the mycoplasma
4 (3/0.8%)
9 (11/3%)
18 (8/2
13 (5/1.3%)
1 H Cofactors, Vitamins, Prosthetic Groups, Pigments (18)
2 Cell Wall and Capsule (4)
3 ■ Virulence, Disease and Defense (14)
4 ■ Potassium metabolism (3)
5 • Photosynthesis (0)
6 ■ Miscellaneous (1)
7 ■ Phages, Prophages, Transposable elements, Plasmids (0)
8 Membrane Transport (11)
9 ■ Iron acquisition and metabolism (0)
10 RNA Metabolism (42)
11 Nucleosides and Nucleotides (11)
12 Protein Metabolism (142)
13 Cell Division and Cell Cycle (5)
14 Motility and Chemotaxis (0)
15 Regulation and Cell signaling (0)
16 Secondary Metabolism (0)
17 DNA Metabolism (47)
18 .IK Fatty Acids, Lipids, and Isoprenoids (8)
19 Nitrogen Metabolism (0)
20 Donmancy and Sporulation (1)
21 Respiration (1)
22 Stress Response (9)
23 Metabolism of Aromatic Compounds (0)
24 Amino Acids and Derivatives (19)
25 Sulfur Metabolism (1)
26 Phosphorus Metabolism (1)
27 Carbohydrates (25)
Fig. 2. The diagram showing the gene attribution to functional groups for the M. hominis MH1866 strain. The picture was acquired using the RAST server. Figures 1-27 are the notation keys for the subsystems in the genome structure. The number of genes in the subsystem / the rate of the genes in the whole genome structure (%) is shown in parenthe-
ses
to derive energy in the form of ATP via an alternative way (arginine and ornithine degradation) [6]. The contribution of the membrane transport products and enzymes involved in the purine metabolism accounts for 3% of the general genome structure.
In every studied mycoplasma isolate, there were genes that encode the efflux systems that participate in membrane transport: namely, ABC transporters (the ATP-binding cassette superfamily) and proteins belonging to MATE (Multidrug and toxic compound extrusion) family). The system of ABC transporters is represented by structural elements performing oli-gopeptide transport via the bacterial cell membrane: namely, by three copies of the oppB gene (encoding the transport proteins of permease OopB) and one copy of the oppC gene (encoding the permease OopC). The function of the efflux pumps of the MATE system is ensured through electrochemical gradient of sodions (Na+) [16]. The length of the whole sequence of the gene encoding proteins that belong to the MATE family in every analyzed mycoplasma strain is 1,809 nucleotides.
As has been stated earlier, the analyzed strains of M. hominis (M45 and M57 MH1866) are characterized by resistance to ciprofloxacin. The search for the mutations responsible for resistance to fluoroquinolones was performed by analyzing the QRDR region in the genes encoding topoisomerases: gyrA and gyrB (DNA gyrase subunits), and parC and parE (topoisomerase IV subunits). Detailed characteristics of the gyrA, gyrB,
parC and parE genes in the M. hominis M45 and M57 isolates were reported in a previously published study [16]. The resistance to ciprofloxacin in the M. hominis M45 and M57 isolates was found to be related to the amino acid substitution of serine (S) for leucine (L) at position 83 in DNA gyrase subunit A [16]. It was discovered that the gyrA, gyrB, parC, and parE genes of the MH1866 isolate contain a great number of nucleo-tide polymorphisms. Thus, 47 point substitutions were found in the gyrA gene; 10 point substitutions, in the gyrB gene; 45 substitutions, in the parC gene; and 19 substitutions, in the parE gene. It was found that the resistance of M. hominis MH1866 to ciprofloxacin is attributable to the mutation in the QRDR region of the parC gene, which leads to an acid substitution of lysine (K) for arginine (R) at position 144 in topoisomerase IV subunit A (Fig. 3).
No meaningful substitutions in the QRDR region of the gyrA, gyrB, and parE genes in M. hominis MH1866 were detected.
The dendrogram of the whole genome nucleotide sequences for the studied strains of M. hominis (M45 and M57 MH1866) with respect to the M. hominis genomes deposited in the GenBank database is presented in Fig. 4.
The results of the phylogenetic analysis showed that the M. hominis M45 isolate holds a separate position with respect to the Russian mycoplasma isolates and constitutes a separate phylogenetic branch. The
Mycoplasma hominis Topoisomerase IV subunit A PG21 (parC) Sequence ID: Query_143601 Length: 933 Number of Matches: 1
Range 1: 1 to 933 Graphics Next Matcn ▼ Previous Matcn ▲
Score 1873 bits (4852)
MH1866 1
PG21 1
MH1866 61
PG21 6 1
MH1866 121
PG21 121
MH1866 181
PG21 181
MH1866 241
PG21 241
MH1866 301
PG21 6 01
Expect 0.0
Method Compositional matrix adjust .
Identities Positives Gaps
926/933(99%) 930/933(99%) 0/933(0%)
MKKDRKEEIQEVTENIIEKNMADIMSDRFGRYSKYIIQQRAIPDARDGLKPVQRRILYSM 60 MKKDRKEEIQEVTENIIEKNMADIMSDRFGRYSKYIIQQRAIPDARDGLKPVQRRILYSM
MKKDRKEEIQEVTENIIEKNMADIMSDRFGRYSKYIIQQRAIPDARDGLKPVQRRILYSM 60
WNLHLKNSEPFKKSARIVGDVIGRYHPHGDSSIYEALVRMAQDWKSNFPLIEMHGNKGSI 120 WNLHLKNSEPFKKSARIVGDVIGRYHPHGDSSIYEALVRMAQDWKSNFPLIEMHGNKGSI
WNLHLKNSEPFKKSARIVGDVIGRYHPHGDSSIYEALVRMAQDWKSNFPLIEMHGNKGSI 120
DDDPAAAMRYTESRLEKISELMLRDLDRKVVKMAPNFDDSEYEPIVLPALFPNLLVNGAK 180 DDDPAAAMRYTESRLEKISELML+DLDRKVVKMAPNFDDSEYEPIVLPALFPNLLVNGAK
DDDPAAAMRYTESRLEKISELMLKDLDRKVVKMAPNFDDSEYEPIVLPALFPNLLVNGAK 180
GIAAGFATEIPPHNLGEVIDATIALIKNPTISIEELSEIVKGPDFPTGAIINGINEIKKA 240 GIAAGFATEIPPHNLGEVIDATIALIKNPTISIEELSEIVKGPDFPTGAIINGINEIKKA
GIAAGFATEIPPHNLGEVIDATIALIKNPTISIEELSEIVKGPDFPTGAIINGINEIKKA 240
LSSGQGRITISSKYHYVYDKKDESKIIGIEIIEIPFGVVKSKLVADIDAIAIDKKISGIK 300 LSSGQGRITISSKYHYVYDKKDESKIIGIEIIEIPFGVVKSKLVADIDAIAIDKKISGIK
LSSGQGRITISSKYHYVYDKKDESKIIGIEIIEIPFGVVKSKLVADIDAIAIDKKISGIK 300
EVLDQTDRNGISIFIQLEDGANADAIIAYLMNKTELSISYSYNMVAIDNNRPVILNLYSA 360 EVLDQTDRNGISIFIQLEDGANADAIIAYLMNKTELSISYSYNMVAIDNNRPVILNLYSA
EVLDQTDRNGISIFIQLEDGANADAIIAYLMNKTELSISYSYNMVAIDNNRPVILNLYSA 360
Fig. 3. Alignment of the amino acid sequence of topoisomerase IV subunit A of the Myco-plasma hominis MH1866 clinical isolate and the reference strain Mycoplasma hominis ATCC 23114 (PG21). Substitution of lysine (K) for arginine (R) at position 144 is shown in red
M. hominis MH1866 isolate is genetically close to the M. hominis MH1817 isolate; together, they form a separate cluster. The M. hominis M57 strain was singled out into a separate branch within the primary group of Russian mycoplasma isolates.
DISCUSSION
Modern molecular methods provide an insight into the functioning of the genome of M. hominis, one of the smallest prokaryote genomes, and allow researchers to trace its evolution. The French group of scholars led by S. Pereyre was the first to decode the full structure of the genome of M. hominis ATCC 23114 (GenBank accession number FP236530.1) in 2009; its size was 665,445 base pairs [6]. As of today (August 23, 2019), the GenBank/NCBI database contains information on the whole genomes of seven M. hominis strains and incomplete genomes of 16 strains in the form of contigs (5 strains) and scaffolds (11 strains). The sizes of the genomes of the studied M. hominis isolates (M45, M57, MH1866) appear to be smaller than those of the mycoplasma genomes deposited in the Gen-Bank/NCBI. However, the results of a bioinformatic analysis presented in Table indicate that the primary characteristics of the genome structure (the number of genes, pseudogenes, RNA, protein coding sequences) of M. hominis strains (M45, M57, MH1866) and the reference strain M. hominis ATCC 23114 are identical. It
should be noted that the data collected in our research agree with the information on other members of the M. hominis species represented in the NCBI Genome database (https://www.ncbi.nlm.nih.gov/genome/ genomes/3075?).
The bioinformatic analysis of the genome structure of M. hominis (M45, M57, MH1866) has made it possible to uncover a great number of pseudogenes. Pseu-dogenes are considered to be a reserve of sequences that recombine with functional paralogous genes and thus ensure their genetic diversity [17]. The number of pseudogenes in the genome structure of the M. hominis M45 and M57 isolates was found to be twice as large as that in the reference strain. It is possible that numerous mycoplasma pseudogenes ensure assortment of sequences, which is required for creating the genetic diversity of surface antigens [17].
The phylogenetic relationships between the studied strains and the strains whose genomes have been deposited in the GenBank database were evaluated by comparing single-nucleotide polymorphisms. The data of the phylogenetic analysis demonstrate that the studied ciprofloxacin-resistant Mycoplasma hominis isolates are genetically heterogeneous. However, a comparative analysis using the RAST server [13] showed a high degree of homology in the protein sequences of the studied mycoplasma strains. A large number of point mutations in the genome of the Mycoplasma
75
100
80
68
100
M. hominis 621 Fig. 4. The dendrogram of
M. hominis strain H34 the whole genome nucle-
M. hominis strain TOA otide sequences for the
M. hominis 1019 Mycoplasma hominis strains
M. hominis 1817 deposited in the internation-
M. hominis 1866 al GenBank/NCBI database.
M. hominis 1002 * denotes the Mycoplasma
M. hominis strain M57 hominis clinical isolate M45
M. hominis 529 that is the farthes from other
M. hominis strain Sprott Russian isolates
M. hominis strain AF1
M. hominis strain AF3
M. hominis ATCC 27545
M. hominis 1991
M. hominis 1861
M. hominis strain M45*
M. hominis strain PL5
M. hominis ATCC 23114
M. hominis strain 387 MHOM 107 14438 1052663
M. hominis strain 403 MHOM 10 5714 76960
hominis strains confer them high genetic plasticity and a tendency toward rapid evolution.
The prevalence of genes encoding proteins with catabolic functions in the Mycoplasma hominis genome is confirmation that the biochemical capacities of mycoplasmas are scarce. Nutrients are supplied into a mycoplasma cell from the host cells predominantly by transport proteins [1]. The transport proteins of mycoplasma are less specific than the transport proteins of other bacteria; they perform several functions. Thus, the OopB and OopC proteins, which are part of the ABC transporter system, not only implement oli-gopeptide transportation, but also participate in drug elimination and excretion out of the bacterial cell [18]. When characterizing the non-specific mechanism of fluoroquinolone resistance in Mycoplasma hominis (M45, M57, MH1866), one should note that the genome of every studied isolate carries a single copy of the gene encoding multiple drug-resistance proteins MATE. The aforementioned gene is multi-component; it contains homologous sequences of the Staphylococcus aureus norM and mepA genes, whose role in the excretion of cationic antimicrobial drugs out of the bacterium cell has been proved [19].
It was determined that the molecular mechanism that ensures fluoroquinolone resistance in the studied Mycoplasma hominis clinical isolates (M45, M57, MH1866) is possibly related to the nucleotide substitutions in the gyrA (S83L) and parC (K144R) genes, which changed the amino acid structure of the proteins of the large subunits of DNA gyrase and topoisomerase IV. This mechanism has been identified in and described for a number of conventional bacteria (E. coli, Streptococcus spp, and Staphylococcus spp.) [20]. The numerous single-nucleotide substitutions in the gyrA,
gyrB, parC, and parE genes of the Mycoplasma hominis isolates account for their high degree of genetic polymorphism and play a crucial role in the formation of AMR, which is consistent with the findings reported in publications [21-23].
CONCLUSIONS
The results of our research revealed a similarity between the genome structure of the ciprofloxacin-re-sistant Mycoplasma hominis (M45, M57, MH1866) clinical isolates, on the one hand, and the reference strain M. hominis ATCC 23114 (GenBank accession number FP236530.1), on the other. We have discovered that genes encoding the proteins involved in catabolic processes are prevalent in the genome structure, which bolsters the aforedescribed theory about the scarcity of biosynthetic capacities in M. hominis [1, 6]. In the genome of the studied mycoplasma clinical isolates, we identified a great number of nucle-otide substitutions that do not affect amino acid codons, which is indicative of their intraspecies genetic and evolutionary diversity. The studied isolates lack resistance determinants with the conjugative transfer mechanism, which explains the dominance of the conventional molecular mechanism of mycoplasma resistance to fluoroquinolones (ciprofloxacin). This mechanism involves mutations in the QRDR region of the gyrA and parC genes. It is possible that the identified genes encoding MATE proteins in M. hominis can lead to excretion of antimicrobial drugs out of the bacterial cell under certain conditions. It is necessary to conduct such research in order both to understand the natural evolution of M. hominis and to gauge the general structure of the urogenital mycoplasma population.
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