Data on the genome and proteome profiles of ciprofloxacin-resistant Acholeplasma laidlawii strains selected under different conditions in vitro

Acholeplasma laidlawii is widespread hypermutable bacteria (class Mollicutes) capable of infecting humans, animals, plants, which is the main contaminant of cell cultures and vaccine preparations. The mechanisms of the development of antimicrobial resistance of this bacterium are associated with the secretion of extracellular vesicles, which can mediate the lateral transfer of antibiotic resistance determinants. We compared the genome profiles of ciprofloxacin-resistant A.laidlawii strains PG8r1 (MIC 10 µg/ml) and PG8r3 (MIC 10 µg/ml) selected under different in vitro conditions - when ciprofloxacin-sensitive (MIC 0.5 µg/ml) A.laidlawii PG8B strain was cultured at increasing concentrations of ciprofloxacin in a broth medium alone, and with vesicles derived from the ciprofloxacin-resistant (MIC 20 µg/ml) A.laidlawii PG8R10c-2 strain, respectively. Genome profiles of PG8c-3 (obtained from a single colony of the strain PG8B) and PG8R10c-2 were analyzed too. Patterns of the quinolone target genes (gyrA, gyrB, parE, parC) containing in extracellular vesicles of PG8c-3, PG8R10c-2, PG8r1 and PG8r3 were determined. Genome sequencing was performed on the NextSeq Illumina platform. Search and annotation of single nucleotide polymorphisms were performed using Samtools and SnpEff, respectively. We also compared cellular proteomes of PG8c-3, PG8r1 and PG8r3. The cellular proteome profiles of the A. laidlawii strains were determined by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. This work presents data on single nucleotide polymorphisms (SNPs) found in the genomes of the ciprofloxacin-resistant strains selected under different in vitro conditions and proteins that were differentially expressed in the cells of ciprofloxacin-resistant strains selected under different conditions in vitro.


a b s t r a c t
Acholeplasma laidlawii is widespread hypermutable bacteria (class Mollicutes) capable of infecting humans, animals, plants, which is the main contaminant of cell cultures and vaccine preparations. The mechanisms of the development of antimicrobial resistance of this bacterium are associated with the secretion of extracellular vesicles, which can mediate the lateral transfer of antibiotic resistance determinants. We compared the genome profiles of ciprofloxacin-resistant A.laidlawii strains PG8r1 (MIC 10 μg/ml) and PG8r3 (MIC 10 μg/ml) selected under different in vitro conditions -when ciprofloxacin-sensitive (MIC 0.5 μg/ml) A.laidlawii PG8B strain was cultured at increasing concentrations of ciprofloxacin in a broth medium alone, and with vesicles derived from the ciprofloxacin-resistant (MIC 20 μg/ml) A.laidlawii PG8R 10 c-2 strain, respectively. Genome profiles of PG8c-3 (obtained from a single colony of the strain PG8B) and PG8R 10 c-2 were analyzed too. Patterns of the quinolone target genes (gyrA, gyrB, parE, parC ) containing in extracellular vesicles of PG8c-3, PG8R 10 c-2, PG8r1 and PG8r3 were determined. Genome sequencing was performed on the NextSeq Illumina platform. Search and annotation of single nucleotide polymorphisms were performed using Samtools and SnpEff, respectively. We also compared cellular proteomes of PG8c-3, PG8r1 and PG8r3. The cellular proteome profiles of the A. laidlawii strains were determined by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. This work presents data on single nucleotide polymorphisms (SNPs) found in the genomes of the ciprofloxacin-resistant strains selected under different in vitro conditions and proteins that were differentially expressed in the cells of ciprofloxacin-resistant strains selected under different conditions in vitro .
© 2020 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) Table   Subject Molecular biology Specific subject area Mollicute genomics and proteomics; antibiotic resistance Type of data A. laidlawii strains with differential susceptibility to ciprofloxacin Description of data collection Three ciprofloxacin-resistant Acholeplasma laidlawii strains were selected under different conditions in vitro : sequentially inoculated in a broth medium that contained increasing concentrations of ciprofloxacin, co-culturing of ciprofloxacin-sensitive strain with and without extracellular vesicles derived from the high-level ciprofloxacin-resistant mollicute. The vesicles of A. laidlawii were obtained by ultracentrifugation; their purity was validated by PCR. Genome sequencing was performed on the NextSeq Illumina platform. Search and annotation of single nucleotide polymorphisms were performed using Samtools and SnpEff, respectively. Cellular proteins were separated by 2DE. Search of differential expressed proteins was performed using PDQuest. Proteins were identified by mass spectrometry.

Value of the Data
• These data document SNPs found in the genomes of ciprofloxacin-resistant Acholeplasma laidlawii strains, which were selected under different conditions in vitro. • These data document proteins that were differentially expressed in the cells of ciprofloxacinresistant A. laidlawii strains, which were selected under different conditions in vitro. • These data may help to investigate different ways of developing ciprofloxacin resistance in A.
laidlawii. • These data may be useful for identification of effective drug targets to eliminate the mycoplasma contamination.

Data Description
This paper presents data on the features of the SNP profiles, as well as a features of patterns of differentially expressed proteins of ciprofloxacin-resistant A. laidlawii strains selected under different conditions in vitro . Genome profiles of the ciprofloxacin-resistant A. laidlawii strains PG8r1 (MIC 10 μg/ml) and PG8r3 (MIC 10 μg/ml) arising respectively when the ciprofloxacinsensitive (MIC 0.5 μg/ml) A. laidlawii PG8B strain was cultured at increasing concentrations of ciprofloxacin in a broth medium alone and with vesicles derived from the ciprofloxacin-resistant (MIC 20 μg/ml) A. laidlawii PG8R 10 c-2 strain, were compared. Genome profiles of PG8c-3 (obtained from a single colony of the strain PG8B) and PG8R 10 c-2 were analyzed too (   teins with the similar character of change are highlighted ( Fig. 4 ). Lists of differentially expressed proteins in ciprofloxacin-resistant A. laidlawii strains are presented in Supplementary Tables 5, 6.

Bacterial strains and culture conditions
Cells of ciprofloxacin-resistant strain A. laidlawii PG8R 10 c-2 (MIC 20 μg/ml) were cultivated in Edward's medium (tryptose 2%; NaCl 0.5%; KCl 0.13%; Tris-base 0.3%; horse blood serum 10%; yeast extract 5%; glucose 1%; penicillin 10 0 0 U/ml; phenol red 0.3 ml of 1% solution) in the presence of ciprofloxacin (10 μg/ml). The minimum inhibitory concentration (MIC) of the cultures was determined using the dilution method in a liquid nutrient medium with various antibiotic concentrations [1] . Cultivation of A. laidlawii PG8B with vesicles of A. laidlawii PG8R 10 c-2 was carried out according to [1] with modifications in triplicates. A. laidlawii PG8B was cultivated in Edward's medium at 37 °C until the middle of the logarithmic phase. Then, the cells were pelleted by centrifugation, suspended in Edward's medium to a concentration of 10 7 cells/ml, and vesicles (15-20 μg/ml protein) isolated from PG8R 10 c-2 strain were added along with ciprofloxacin at a concentration of 0.5 μg/ml. Suspensions were incubated for 6 h at 37 °C, then Edward's medium was added to each suspension and they were incubated at 37 °C until the middle of the log phase. This scheme was repeated, with an increase in the concentration of the antibiotic in each cycle, to ciprofloxacin concentration of 10 μg/ml. The ciprofloxacin-resistant (MIC 10 μg/ml) strain resulting from this procedure was designated PG8r3. The ciprofloxacinresistant (MIC 10 μg/ml) PG8r1 strain was obtained similarly, but without the addition of PG8R 10 c-2 vesicles. Patterns of the quinolone target genes (gyrA, gyrB, parE, parC ) containing in extracellular vesicles of PG8c-3, PG8R 10 c-2, PG8r1 and PG8r3 were determined (Supplementary Table 1).

Extraction and purification of extracellular vesicles
The isolation of the A. laidlawii PG8R 10 c-2 extracellular vesicles was performed according to [2] . The cells were removed from the culture broth by centrifuging at 60 0 0 g for 20 min, after which any residual cells were removed from the supernatant by filtration using 0.1 μm PES filter (Sartorius). Supernatant was concentrated using 100 kDa Vivacell 100 (Sartorius, Germany). The vesicles were pelleted by ultracentrifugation at 10 0,0 0 0 g, 1 h, 8 °C (Beckman Coulter Optima TM MAX-E). Crude EVs preparation was then resuspended in buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM MgCl 2 ) and placed on a stepwise density gradient 20%-40% Optiprep (Sigma) and ultracentrifuged (10 0,0 0 0 g, 3 h, 8 °C). The vesicular fraction was collected, diluted threefold in buffer and then ultracentrifuged again. The pellet was resuspended in buffer supplemented with 1 mM PMSF (Fluka) and stored at 8 °C. The absence of microbial cells in the vesicle preparation was tested, plating on Edward's medium and PCR analysis with primers for marker nucleotide sequences of vesicle -16S-23S rRNA gene intergenic spacer region − , ftsZ − , pnp + , tufB + .

DNA preparation and sequencing
DNA was isolated from the cells of strains ( A. laidlawii PG8Bc-3, PG8R 10 c-2, PG8r1, and PG8r3), using the phenol extraction method with additional treatment with proteinase K and RNase [3] . Whole genome sequencing was performed on the NextSeq Illumina platform (USA). DNA was fragmented enzymatically using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina. Libraries were created from the obtained DNA fragments according to the manufacturer's instructions. The quality of the resulting libraries was evaluated using a 2100 Bioanalyzer instrument (Agilent Technologies). DNA concentration was determined using a Qubit 2.0 fluorimeter (Invitrogen).

Proteins preparation
Proteins from cells of A. laidlawii were isolated as described previously [2] . The A. laidlawii cells were pelleted (60 0 0 g, 20 min) and washed twice with buffer (150 mM NaCl, 50 mM Tris, 2 mM MgCl 2 .6H 2 O, pH 7.4) and once in the same buffer with PMSF. The pellet of cells was treated with CHAPS and Micrococcal Nuclease Mix (Thermo Fisher Scientific, USA). The resulting proteins were dissolved in a solution containing 8 M urea, 2 M thiourea, 5% ampholines (pH 3-10), 80 mM dithiothreitol (DTT), 5% CHAPS and 1.67% NP-40. The protein concentration in the samples was measured by the Bradford method.

Tryptic digestion of proteins
Proteins were extracted from the gel and hydrolyzed using the protocol described in [6] . The protein spots were cut out from the gel and washed in ddiH 2 O and mix of acetonitrile: 200 mM NH 4 HCO 3 (1:1) at 50 °C 30 min. Protein reduction was performed using 10 mM DTT 100 mM NH 4 HCO 3 for 1 h, followed by alkylation using a mixture of 50 mM iodoacetamide and 100 mM NH 4 HCO 3 in the dark for 45 min at room temperature. The gels were incubated in acetonitrile, dried and incubated in trypsin Gold (Promega) solution for 60 min at 4 °C. Tryptic digestion was performed at 37 °C overnight. To extract peptides, a solution containing 0.1% trifluoroacetic acid in deionized water was added to gel fragments that were then incubated in an ultrasonic bath for 10 min. The resulting supernatants were sampled into separate tubes.

Protein identification by MALDI TOF/TOF MS
The identification of proteins was performed using MALDI-TOF/TOF mass spectrometer Ultraflex III BRUKER (USA) with a UV-laser in the positive ion mode in the diapason of 50 0-40 0 0 Da using reflectron [7] . The proteins were identified from the masses of proteolytic fragments using Mascot Peptide Mass Fingerprint (Matrix Science, USA) software and UniProt database. Searches were performed allowing up to 1 trypsin miscleavage. Variable modifications included carbamidomethylation of cysteine and oxidation of methionine. Peptide mass tolerance was set to 60 ppm. A protein score of ≥44 was considered a significant matched ( p < 0.05).

Ethics Statement
The work involved bacteria, but did not involve the use of human subjects or animals.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.