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MOLECULAR-GENETIC CHARACTERISTICS
OF M. HOMINIS CLINICAL ISOLATES EFFLUX SYSTEMS
E.A. Kolesnikova1*, N.F. Brusnigina1, M.A. Makhova1, A.E. Alekseeva1, E.V. Pligina2
1 Academician IN Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology, Federal Service for Surveillance on Customers Rights Protection and Human Wellbeing, 71 Mallaya Yamskay st., Nizhny Novgorod, 603950, Russia;
2 Federal State Budgetary Educational Institution of Higher Education «Privolzhsky Research Medical University» of the Ministry of Health of the Russian Federation, 10/1 Minin & Pozharsky sq., Nizhny Novgorod, 603005, Russia.
* Corresponding author: shmelevael@yandex.ru
Abstract. The widespread occurrence of drug-resistant urogenital mycoplasmas and the high rate of their genetic variability make it possible to consider Mycoplasma hominis as a natural reservoir of antibiotic resistance determinants. Multiple drug resistance of many bacterial types is due to the active elimination of the antibiotic from the cell through various efflux systems. The genome structure of ten clinical isolates Mycoplasma hominis was studied using next-generation sequencing. The molecular genetic characterization family MATE proteins of Russian isolates M. hominis genes using bioinformatics analysis was obtained in the first. It has been established that proteins of the transport systems (ABC-transporters) and MATE play an important role in the formation of antibiotic resistance in urogenital mycoplasmas. The activity and functioning of transport systems are associated with the presence of mutations in the genes encoding regulatory proteins. Efflux systems should be considered as promising potential targets in the creation of new generation antibacterial drugs.
Keywords: Mycoplasma hominis, antibiotic resistance mechanisms, efflux system, ABC transporters, MATE.
List of Abbreviations
ABC transporters - ATP-binding cassette superfamily
BLAST - Basic Local Alignment Search Tool
CARD - Comprehensive Antibiotic Resistance Database
DNA - Deoxyribonucleic Acid MATE - Multidrug and toxic compound extrusion family
MDR - Multiple drug resistance MFS - Major Facilitator Superfamily NCBI - National Center for Biotechnology Information
NGS - Next Generations Sequencing PGAP - Prokaryotic Genome Annotation Pipeline
QRDR - Quinolone Resistance-Determining Region
RAST - Rapid Annotation Subsystem Technology
RND - Resistance Nodulation Division SMR - Small Multidrug Resistance UGT - Urogenital Tract
Introduction
Representative class Mollicutes Mycoplasma hominis is the smallest currently known prokaryote and refers to opportunistic microorganisms (Borchsenius et al., 2016). At realizing its pathogenic properties, Mycoplasma hominis can cause inflammatory diseases of the urogenital tract (UGT), complications of pregnancy and childbirth, as well as infectious diseases of newborns (Belova & Nikonov, 2015; Moridi et al. , 2020). Treatment of urogenital infections associated with M. hominis is often difficult, due to the natural resistance of the bacterium to drugs that inhibit cell wall synthesis (Sidorenko & Tishkov, 2004). According to the literature, recently, there has been an increase in the number of M. hominis isolates resistant to fluoroquinolones, macrolides, tetracyclines, which are most often used in the treatment of inflammatory diseases (Chernova et al., 2016). Long-term, asymptomatic persistence of Mycoplasma hominis isolates in the host organism, and uncontrolled use of antibiotics lead to selective pressure and the emergence of multi-resistant mycoplasma strains (Kolesnikova et al., 2018).
The widespread occurrence of drug resistant urogenital mycoplasmas, the high rate of their genetic variability allows us to consider this microorganism as a natural source (reservoir) of antibiotic resistance determinants (Kolesnikova et al., 2019). There are several mechanisms in the bacterial cell that provide antibiotic resistance, including the degradation of drugs, modification of the targets of antibiotic action, as well as the emergence of alternative pathways, and the elimination of antibiotics from the cell (Chernova et al., 2016). Efflux systems, belonging to the families of ABC transporters (ATP Binding Cassette), RND (Resistance-Nodulation Division), MFS (Major Facilitator Superfamily), SMR (Small Multidrug Resistance), and MATE (Multidrug and Toxin Extrusion) play an important role in the emergence of multidrug resistance in many species of gram-positive and gram-negative bacteria (Fernandez &. Hancock, 2012; Sun et al., 2014). Search and molecular characterization of the nature and characteristics of efflux systems of bacteria, including Mycoplasma homi-nis, is extremely difficult when using classical microbiological research methods. In scientific research, the advent and widespread use of modern molecular genetic methods, including high-throughput sequencing (NGS), have made it possible to deeply study the structural organization of bacterial genomes, including a detailed study of efflux systems.
Materials and Methods
The study subjects were ten M. hominis clinical isolates (M45, M57, MH1866, MH1817, MH529, MH1002, MH621, MH1019, MH1861, MH1991), 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. Detection, identification, determination of anti-bioticogram to seven antibacterial drugs (doxycycline, gentamycin, ofloxacin and ciprofloxacin, clindamycin, midecamycin and josamycin) mycoplasmas were carried out using commercial liquid differential diagnostic media produced by the Central Research Insti-
tute of Electrical Engineering of Rospotrebnad-zor (registration number FSR 2008/03366). DNA study was isolated and purified sorption method using a commercial kit AmpliPrime DNA-sorb-V (2012/14019). The DNA concentration in the samples was determined using a Qubit fluorometer (Invitrogen, Austria). The DNA library for sequencing was prepared using the Nextera XT kit (Illumina). The quality of the DNA library was assessed on a QIAxel advanced system (Qiagen, Germany) using a QI-Axcel DNA Fast Analysis Kit (15 - 3000 bp) for separating DNA fragments.
Whole genome sequencing was performed on a MiSeq sequencer (Illumina, USA) using the MiSeq reagent kit v2 (Illumina) for 500 cycles. The whole genome sequence of the M. hominis strain ATCC 23114 (GenBank FP236530.1) was chosen as a reference. Read archives and nucleotide sequences of whole genomes of 10 clinical isolates of M. hominis are available in the GenBank / EMBL / DDBJ database. The nucleotide sequences were aligned using the MiSeq sequencer firmware (Isis version 2.6.2.3).
Visualization and analysis of the obtained data were performed using the UGENE Unipro 1.31 software. Genome annotation was performed using a Rapid Annotation using Subsystem Technology (RAST) (Aziz et al., 2008) and NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Sequence alignment was performed using the CLUSTALX 2.0 software (Thompson et al., 1994). Sequence analysis of genes encoding ABC transporter proteins and MATE isolates Mycoplasma hominis carried out using BLAST algorithm and software package presented on the NCBI server. Comparison and Search antibiotic resistance was performed using reference DNA and protein sequences placed in a complex database (CARD) (Alcock et al., 2020).
Homologous protein tertiary structure model MATEobtained using Phyre2 web portal for protein modeling server and UGENE Unipro 1.31 software. Phylogenetic trees were built with the neighbor-joining algorithm (Okonech-nikov et al., 2012) in the MEGA7.0 software 6.0 (Kumar et al., 2016).
Results
The resistance profile of the studied strains M. hominis was determined: three strains (M45, M57, MH1866) were characterized by resistance to ciprofloxacin, three strains (MH1019, MH1861, MH1991) - to midecamycin, one strain MH1002 - to ofloxacin, strain MN529 was resistant to ciprofloxacin and midecamycin, isolate MH1817 was susceptible to all drugs included in the study. Molecular genetic characterization, the of the genome of all the strains under study was obtained using NGS sequencing. The whole-genome nu-cleotide sequences of M. hominis have been deposited in the international data base NCBI GenBank under the accession numbers MRAY00000000 (M. hominis М45), MRAX00000000 (M. hominis М57), QNHJ00000000 (M. hominis MH621), Q0K000000000 (M hominis MH1866), Q0KP00000000 (M. hominis MH1991), QNHH00000000 (M hominis MH1019), Q0KQ00000000 (m hominis MH1817), QNHI00000000 (M. hominis MH1002), QNHJ00000000 (M. hominis MH621), Q0KN00000000 (M hominis MH1861), QMJZ00000000 (M. hominis MH529). Bioinformatic analysis of the nucleotide sequence of the genome clinical isolates M. hominis have been allowed discover genes encoding efflux systems involved in membrane transport and active elimination of antibiotics from a bacterial cell, namely ABC transporters (ATP-bind-ing cassette superfamily) and proteins of the MATE family (Multidrug and toxic compound extrusion family). An analysis the data acquired using the RAST server, showed that the ABC transporter system of the M. hominis isolates under study is represented by structural elements that transport oligopeptides through the bacterial cell membrane, determined by three copies of the oppB gene (encodes transport proteins -0opB permeases) and one copy of the oppC gene (encodes 0opC permease).
Molecular genetic characteristics of the MATE protein family of Mycoplasma hominis clinical isolates. To in addition to ABC transporters, one copy of the gene encoding a protein ho-
mologous to proteins from the MATE family of multiple drug resistance was found in the genome of all M. hominis isolates under study (Multidrug and toxic compound extrusion family). The analysis of the profile of the gene encoding proteins of the MATE family revealed that in nine strains the length of its nucleotide sequence was 1809 nucleotides, and the amino acid sequence was 602 amino acids. The length of the gene encoding the MATE protein in the MH529 isolate was longer than in every analyzed M. hominis strain included and amounted to 1998 nucleotides and 665 amino acids. The tertiary structure (3D model) of the MATE protein of the clinical isolate MH529 is shown in Figure 1.
It was determined that the genes encoding proteins of the MATE family in the studied M. hominis isolates do not belong to the classic endogenous pumps of the active efflux system, such as AcrB in E.coli (Raherison et al., 2005), MexB in Pseudomonas aeruginosa, QacA in Staphylococcus aureus (Kaatz et al., 2005) and PmrA in Streptococcus pneumonia (Fernandez et al.,2012). Mycoplasma genes encoding MATE are multicomponent and contain incomplete homologous sequences of two families: MATE_like superfamily (MATE_like 5, 8, 4, 14, 6, MATE_MeA_like, MATE_yoeA_like) and NorM superfamily (vmrA, NorM, matE)
(Fig. 2).
Comparative analysis of gene sequence, family proteins encoding MATE ten M. homi-nis strains relative to the reference strain (GenBank number FP236530.1) has been revealed significant differences in the number of nucleotide substitutions (from 30 to 49) (Fig. 3). At the same time point mutations leading to a change in amino acid codons were much less common: from one in M. hominis M45 to four in every from analyzed strain M. hominis MH1817, M. hominis MH1019, M. hominis M57.
According to the CARD 2020 database, the genes encoding the MATE proteins of M. hom-inis are most similar to the genes responsible for multidrug resistance, namely: cdeA Clostridioi-des difficille (Dridi et al., 2004), mepA Staphylococcus aureus (Kaatz et al., 2006) and norM Pseudomonas poae. The mepA gene encodes a protein MepA, which is part of the MepRAB
73
A
Image of the tertiary structure of the MATE protein (UGENE Unipro 1.31)
B
Color Image N ^ C - Terminal End Model dimensions (Â):X: 61.839 Y: 63.855 Z: 67.575
Fig. 1. Tertiary image of the protein structure of the MATE system in the clinical isolate M. hominis MH529. Images were obtained by the Phyre2 web portal for protein modeling server (A, B) and UGENE Unipro 1.31 software (A)
Fig. 2. Characterization of the structure of genes family proteins encoding MATE on the example of M. hominis clinical isolate MH529
GenBank FASTA
Link To This View 1 Feedback
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114 see 114,900 IIS К 15,100 115,200 115,309 115,400 115,500 115,600 115,700 115.800 115,900 116 К 116,10 116,200 116,300 116,400 116,500 116,600 116,700 116,800 116,900
Genes ULI ULI I1HOJ150 к
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QueryJ5541
(liieryJ5542
Queryj5543
QueryJ5544
Queryj5545
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114 800 114,900 115 К 115,100 115,200 115,300 115,400 115,500 115,600 |15,700 115.800 |15,900 |16 К 116,10 116,200 jl6,300 116,400 |16,500 116,800 116,700 |16,800 116,900
FP236530.1:15K..17K(2,2Kbp) 0 Tracks shown: 4/10
Fig. 3. Comparative analysis of the sequence of genes, family proteins encoding MATE ten isolates M. hominis relative to the reference strain (GenBank number FP236530.1)
gb|AY661734.1|+| 840-2195|mepA [Staphylococcus aureus] gb IAJ574887.11 -1371-16961 cdeA [Clostridioides difficile]
fig 16666666.220762.peg.219 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis M45] fig 16666666.201005.peg.506 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis M57] fig 12098.26.peg. 132 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis 1019] fig|2098.28.peg.271 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis 1861] fig 12098.27.peg.296 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis 1817] figl 2098.24.peg.15 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis 621] fig 12098.29.peg. 198 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis 1866] figl 2098.23.peg. 180 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis 529] figl 2098.25.peg. 167 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis 1002] figl 2098.30.peg. 183 Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of MDR efflux pumps [Mycoplasma hominis 1991] NZ_LT629706.1: c3787785-3786409 Pseudomonas poae, штамм LMG 21465, хромосома I
Fig. 4. The dendrogram of the genes' sequences annotated in GeneBank/NCBI encoding MATE family proteins of M. hominis strains (MH45, MH57, MH529, MH621, MH1002, MH1019, MH1817, MH1861, MH1866, MH1991) and Pseudomonas poae, Clostridioides difficiUe, Staphylococcus aureus. Phylogenetic tree was constructed by the UGENE Unipro software
cluster, it is involved in the clearance of drugs from the bacterial cell (Kaatz et al., 2006).
In order to determine the evolutionary origin of the MATE system proteins of M. hominis isolates, a phylogenetic analysis of the relationship with the most homologous proteins re-sponsible for the multiple drug resistance of bacteria other species was carried out (Fig. 4).
The high level of homology of genes encoding MATE proteins in all studied M. hominis strains by phylogenetic analysis was indicated. The highest degree of gene homology is observed in pairs of clinical isolates that form single clusters with each other. M. hominis MH1002 - M. hominis MH1991, M. hominis MH1866 -M. hominis MH621 and M. hominis MH1019 - M. hominis MH1861.
However, the genes sequences in M. hominis M45 and M. hominis M57 stains are the most distant from the general studied genes group, they are isolated as a separate phylogenetic branch. The genes sequences encoding the MATE proteins in multiresistant M. hominis MH529 strain and sensitive to antibiotics M. hominis MH1817 strain do not form single clusters with other strains. They are located separately, but within the general group of the studied mycoplasmas.
Discussion
It is known that the efflux systems of ABC transporters are evolutionarily conservative, they are able to export a large number of biological compounds due to the energy of ATP hydrolysis (Raherison et al., 2005). This property provides bacteria resistance not only to various classes of antibiotics, but also to disinfectants. Thus, proteins OopB and OopC, which are the part of the ABC transporter system in M. hominis, are involved not only in the transport of oligopeptides, but also in the removal of drugs from the bacterial cell. To date, the role of efflux in the development of resistance to fluoroquinolones by ABC transporters has been experimentally vindicated (Fernandez et al., 2012; Sun et al., 2014). The data of ciprofloxacin-resistance of M. hominis isolates M45 M57, MH1866, caused by muta-tional changes in genes encoding topoisomer-ases, were reported in a previously published study (Kolesnikova et al., 2018).
We did not find mutations in the «hot» spots of the gyrA/gyrB and parC/parE genes in the M. hominis MH1002 isolate. Probably, the resistance to ofloxacin of the studied M. hominis strain MH1002 is associated with the active antibiotic elimination from the bacterial cell by work of ABC transporters.
It has been shown that the studied MATE efflux system genes of clinical mycoplasma isolates were identical to genes encoding proteins of the MATE family in representatives Eubac-terium and Clostridium (Eubacterium ventrio-sum WP 005361628.1; Clostridium tetani E88
WP_017415026.1, Clostridioides difficille OGO77753.1) on 20-27%. The role of these proteins in the development of multidrug resistance is well established (Dridi et al., 2004).
Proteins of the MATE family of M. hominis clinical isolates are similar to the integral membrane proteins - MepA of Staphylococcus aureus; they are involved in the export of metabolites across the cell membrane and also responsible for multidrug resistance (MDR) of various species bacteria. So, in the works of Chen et al. (2002) on the example of Vibrio parahaemolyt-icus, Vibrio vulnificus and E. coli had been proved, that the genes vmrA and NorM are Na+-antitransporters of drugs in the MATE efflux system and associated with multiple drug resistance (Sun et al., 2014).
As we have shown, the genes encoding proteins of the MATE family in nine Mycoplasma studied isolates were characterized by the same size: 1809 nucleotides. However, the length of the gene encoding the MATE protein in M. hominis isolate MH529, resistant to ciprofloxacin and midecamycin, was significantly longer and amounted to 1998 nucleotide bases. Changes in the QRDR region of the gyrA/gyrB and parC/parE genes in isolate MH529 was not detected, which probably indicates the active removal of antibiotics from the bacterial cell by efflux system MATE.
The gene encoding the protein of the MATE system in M. hominis isolate M45 is phyloge-netically distant from similar genes of other studied mycoplasmas, it is closer to the gene cdeA Clostridioides difficille; its role in the development of fluoroquinolone resistance is well established (Dridi et al., 2004). There is no information about the MATE efflux systems urogenital mycoplasmas characterization in the public domain. It is makes difficult to conduct in-depth comparative analysis. The wide representation mycoplasmas genome circulating in different countries of the world in the international GenBank/NCBI database will further allow assessing the structural organization of the main types efflux systems involved in the multi drug resistance emergence.
Conclusion
To sum up, detailed characteristic of genes encoding efflux system MATE Russian M. hominis isolates was obtained in first. It has been established that proteins of the efflux systems ABC -transporters and MATE play an essential role in the formation of antibiotic resistance in urogenital mycoplasmas. It has been shown that the activity and functioning of transport systems were associated with the presence of mutations in the genes
encoding regulatory proteins. The efflux systems should be considered as promising potential targets in the creation of new generation antibacterial drugs.
Acknowledgements
The reported study was funded by State Task of the Federal Service for Surveillance on Customers Rights Protection and Human Wellbeing the Russian Federation № 121092300067-5.
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