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INTRODUCTION
Poultry production is one of the main food industries worldwide because of its contribution to feeding rapidly growing human populations, low production costs and the absence of cultural and religious restrictions on its consumption (1). In Ecuador, poultry production represents one of the most important industries and it is a source of income for small farmers (2). Mycoplasma gallisepticum causes a chronic respiratory disease in chickens (3), which can lead to their slaughter. It also causes a decrease in weight gain, meat and egg production efficiency (3, 4, 5). Fluoroquinolones (FQs) are broad-spectrum antibiotics that are widely used for the treatment of different diseases in animals, including M. gallisepticum (6). The emergence of resistance to FQs is primarily due to point mutations resulting in amino acid substitutions within the quinolone resistance-determining regions (QRDRs) of the DNA gyrase subunits GyrA and GyrB and/or topoisomerase IV ParC and ParE subunits in bacterial species, including mycoplasmas (7,8). The central mechanism involves alterations of the GyrA subunit and/or the ParC subunit, whereas alterations in GyrB and ParE play a complementary role (7).
Enrofloxacin, oxytetracycline and tylosin resulted in the highest number of resistant isolates of M. gallisepticum in most geographic distributions (9). Surveillance of antimicrobial resistance in clinical strains of M. gallisepticum is essential for determining subsequent treatment guidelines. However, isolation of M. gallisepticum in culture remains a labor-intensive and time-consuming task (10). Mycoplasmas are slow-growing microorganisms with complex requirements. Consequently, standard procedures used for susceptibility testing of classical bacteria, such as disk diffusion method or minimum inhibitory concentration (MIC), are not routinely recommended but only performed by specialized laboratories (9,11). Therefore, to assess potential resistance to fluoroquinolones, a molecular approach that does not involve culture and in vitro antimicrobial susceptibility testing will be applied. The aim of this study was to determine the occurrence of mutations in the quinolone resistance-determining regions (QRDRs) of the genes gyrA and parC in M. gallisepticum positive field samples from broiler flocks in Ecuador.
MATERIALS AND METHODS
The study included a total of 24 M. gallisepticum PCR-positive field samples collected during 2018 and 2019 from 22 commercially reared broiler flocks located in different areas of Manabí province, Ecuador. Bacterial DNA extracts used as templates were prepared by the heat boiling method described by Hernández et al. (12). The samples were previously evaluated and confirmed as positive by PCR for M. gallisepticum, as described by De la Cruz et al. (13). QRDRs were amplified using gene-specific primers designed from the genomic sequence of M. gallisepticum strain R (accession no. AE015450), which included gyrA-F5'-GAGCTAGAAACATCATTCATGG-3' and gyrA-R 5'-CCTACAGCAATACCACTT GAA-3' for the gyrA gene, and parC-F 5'-GATCTTGATGATATATCGTCAC-3' and parC-F 5'-CCAGTTGAACCATTAACGAGT-3' for the parC gene (14). PCR reactions were performed in a total volume of 50 μl containing 1× GoTaq® Green Master Mix (Promega®, Madison, WI, USA), 800 nM of each primer; and 100 ng/5μL of positive mycoplasma sample were used as DNA template. All PCR amplifications were performed on an Eppendorf Mastercycler Gradient Thermal Cycler (Eppendorf, Hamburg, Germany). Nuclease-free water was used as a negative control. DNA from M. gallisepticum strain K3254, code 6/85, was used as a positive control. Amplification products were submitted to electrophoresis on 1.5 % agarose gels. Gels were stained with ethidium bromide (0.5 mg/mL). A 100 bp DNA ladder (Promega®, Madison, WI, USA) was used as molecular weight marker.
To analyze the genetic characteristics of the QRDR, PCR products from six M. gallisepticum-positive samples (AVMG1 to AVMG6) were selected according to their origin in the northern, southern and central region of Manabí. Amplicons were purified using the QIAquick Gel Extraction kit (Qiagen, Santa Clarita, CA, USA), according to the manufacturer's instructions, and sent for direct sequencing at the Macrogen facility (Macrogen Inc. Company, South Korea). Raw sequences were assembled and edited using Geneious R11 v11.0.3 software (Biomatters Limited, Auckland, New Zealand). The sequences obtained were identified by checking, using the BLASTn search program (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Nucleotide sequences were aligned with MAFFT v.7 configured for maximum accuracy (MAFFT with default settings) (15). Theoretical translation of nucleotide sequences to amino acid sequences was carried out on the ExPASy molecular biology web server (http://www.expasy.org). Protein sequences were aligned using ClustalW, included in the BioEdit v.7.0.0 package (Tom Hall Ibis Biosciences, USA).Sequences were sent to the GenBank database with accession numbers MK210575-79 for gyrA and MK210580-83 for parC. For sequencing analysis, substitutions were noted as follows: Xxx##Yyy, where Xxx represents the wild-type amino acid, ## the codon number, and Yyy the substituted amino acid. E. coli gene sequence numbering system was used (16).
RESULTS AND DISCUSSION
Mycoplasma spp. are not affected by common antibiotics that target cell wall synthesis; therefore, other antimicrobials such as tetracycline, macrolides and fluoroquinolone are required (1,9, 14). The main objective of this study was to determine the occurrence of mutations related to acquired resistance to FQ in the gyrA and parC genes corresponding to the DNA gyrase and topoisomerase IV subunits in MG-positive samples from poultry farms in Ecuador. Both enzymes are essential for bacterial DNA replication, thus these genes are found in all strains and are targets for resistance mutations to FQ (17). Hence, as expected, gyrA and parC genes were amplified from a total of twenty-four field samples, and the amplicons corresponded to the expected size of 484 bp and 463 bp for gyrA and parC genes, respectively.
Mycoplasmas are slow-growing microorganisms with complex requirements. Consequently, standard procedures used for susceptibility testing of classical bacteria, such as disk diffusion method or minimum inhibitory concentration (MIC), are not routinely recommended but only performed by specialized laboratories (6, 9). This study was limited by the lack of MG strains to assess microbial susceptibility by phenotypic testing, which would allow defining the MIC of FQ and corroborating the effects of resistance mutations. In this context, the screening of key genetic mutations directly from clinical samples by PCR and direct DNA sequencing is a well-established method, which could be very useful in many laboratories due to the fastidious nature of mycoplasmas. However, some difficulties may arise in the interpretation of DNA chromatograms resulting from direct sequencing of polybacterial samples. That is the reason why, although advantageous, it is still a challenge to optimize DNA extraction, PCR and DNA sequencing directly from the polybacterial sample, such as that used in this work (18). For this reason, even when both genes were amplified from all 24 samples, it was not possible to recover enough DNA from all the amplicons that were purified for sequencing and, therefore, only five sequences were obtained for the gene gyrA and four for the gene parC.
The DNA sequences obtained for gyrA with the following accession numbers MK210575-79 were derived from specimens AVMG2, AVMG3, AVMG4, AVMG5, and AVMG6, respectively. The sequences were highly identical to each other (98, 97-100 %) and >99 %, the same as M. gallisepticum strain S6 (accession number NC023030.2) (Figure S1). The gyrA gene sequences obtained showed eight nucleotide substitutions, most of which were silent mutations (5/8; 62.5 %). Sample AVMG2 (accession number MK210575) revealed seven differences in nucleotide sequence compared to the reference strain M. gallisepticum S6 representing two transverse and six transitional changes. Three substitutions resulting in an amino acid change were detected in all samples, which included replacements at positions 83 (Serine ATT→ Isoleucine AGT) and 157 (Isoleucine ATT→ValineGTT), while 59 (Histidine CAT→ Tyrosine TAT) was only found in sample AVMG2 (E. coli numbering) (Table 1).
There are some studies on the molecular mechanism of FQ resistance in bacteria that have been used E. coli sequence numbering system as a reference. The E. coli numbering system used in this study showed that, for MG, the start codon was located at the tenth amino acid; therefore, the amino acids at positions 68, 69, 93 and 97 in MG according to the E. coli numbering system corresponded to positions 58, 59, 83 and 87 in E. coli. However, some authors did not specify the numbering system used, resulting in discrepancies regarding the location of the resistance mutations (Figure 1). The use of different numbering systems made difficult to confirm previously described mutations and to clearly identify new mutations (20,21).
Previous studies carried out in other bacteria and mycoplasmas have reported quinolone-resistant hot spots by substitution at positions 59, 83, and 157 of GyrA described in M. gallisepticum field samples from Ecuador (18,19). Lysnyansky et al. (13) described that genotype changes at position 59 of GyrA favored susceptibility to enrofloxacin, whereas Sahar and Abou-Khadra (20) reported a fluoroquinolone-resistant isolate of M. gallisepticum that had an amino acid substitution at that position. This may have also been attributed to the presence of additional mutation sites. However, Ser-83→Ile mutation has been correlated with fluoroquinolone-resistant mutants of M. gallisepticum, which showed increased MIC in vitro (22). Substitutions at position 83 in GyrA were highly variable (e.g., Ser-83→Leu/Trp/Phe/Tyr/Asn/Arg), where some mutations caused greater increases in resistance than other substitutions in different bacteria and mycoplasmas due to structural differences between amino acids (23, 24,25).
The analysis of the nucleotide sequences obtained for the parC-amplified 463 bp gene fragments was only possible from four M. gallisepticum field samples (i.e. AVMG1, AVMG2, AVMG4 and AVMG6) with corresponding accession numbers (MK210580-Mk210583), respectively. The sequences were 100 % identical to each other and 98.35 % identical to the M. gallisepticum strain (accession number NC004829.2). The parC gene sequences obtained in Ecuador revealed eight nucleotide substitutions compared to the reference strain M. gallisepticum S6, resulting in one transverse and seven transient changes (Figure 2). Most of the nucleotide substitutions were silent mutations (7/8; 91.67 %), resulting in amino acid substitution at position 80 (Serine TTA→ Tryptophan TGA) in all samples (E. coli numbering) (Table 1). Amino acid changes in parC QRDRs played a key role in the development of fluoroquinolone resistance, which were generally detected at positions 80 and 84 (E. coli numbering) (19, 20, 25). Serine replacement was the most common in conferring quinolone resistance. Mutations at that residue generally had little effect on the catalytic activity of DNA gyrase and topoisomerase IV (6, 25).Interestingly, the most reported mutation at position 80 of parC for M. gallisepticum clinical strains was Ser-80→Leu, whereas Ser-80→Trp replacement was only detected in the Mycoplasma gallisepticum enrofloxacin-resistant mutant obtained by serial passages in vitro (23).
Unlike other bacteria, the horizontal gene transfer (HGT) of mobile genetic elements (MGE) carrying antimicrobial resistant genes (AMR) has been little studied on mycoplasmas. Hence, the main pathway described for the emergence of AMR in Mycoplasma spp. is the occurrence, selection, and fixation of chromosomal mutations in target genes, mainly for synthetic antibiotics such as FQ (26). In addition, mycoplasmas are characterized by a high mutation frequency related to their limited amount of genetic information related to the SOS response and DNA repair systems (9).
To summarize, the results revealed that, in the samples from Ecuador, the following mutations Ser-83→Ile in GyrA and Ser -80→Trp in ParC were found simultaneously. Although the effects of amino acid replacements in DNA gyrase and topoisomerase IV in fluoroquinolone-resistant M. gallisepticum isolates are not fully elucidated, some studies have described the association between MIC results by in vitro microbial susceptibility testing and mutations in QRDRs (20,22, 23). Amino acid change also reveals an important influence on the degree of resistance. For example, MIC values between 0.1-0.025 µg/ml are reported in bacterial strains harboring no mutations (14). An analysis of different results in strains harboring the mutations described here showed that Ser-83→Ile in GyrA was sufficient to reach a level of resistance to enrofloxacin in vitro with MIC≥2 μg/ml, whereas the Ser-80→Trp change detected in ParC, had a greater impact on the level of resistance with a 16-fold increase (MIC≥16 μg/ml) (20, 22).
The contribution of mycoplasmas to the global gene flow associated to resistance among different genera of bacteria is low, as the genomic support of resistance is essentially point chromosomal mutations (27). However, the use of antimicrobials to control mycoplasma infections in both animals and humans requires the support of laboratory testing, as these compounds affect other bacterial genera that share the same niches, contributing to the selection of resistant strains. Besides, the use of antimicrobials for their control without the support of laboratory tests also affects other genera of bacteria present in mucous membranes, facilitating the selection of resistant strains.
Up to date, this is the first study carried out in Ecuador focused on the molecular characterization of QRDRs in genes encoding the synthesis of DNA gyrase and topoisomerase IV enzymes, from field samples of M. gallisepticum collected from broiler chickens. The alterations found in the genes studied have been identified by other authors in previous studies with an increase in MIC values in vitro susceptibility tests using mutants of different Mycoplasma spp. species, including M. gallisepticum (28, 29).These findings could suggest that M. gallisepticum clinical strains with lower susceptibility to fluoroquinolones may have emerged from poultry farms in Manabí province, Ecuador. For this reason, although genotypic methods are advantageous for earlier detection of potential resistance candidates, further studies are needed to culture clinical strains of M. gallisepticum and confirm the possible influence of these substitutions on FQ susceptibility in the genes using antimicrobial susceptibility testing.
Table 1 Amino acid changes in GyrA and ParC QRDR fragments in Mycoplasma gallisepticum positive samples from chickens in Ecuador. / Cambios de aminoácidos en los fragmentos QRDR de GyrA y ParC en muestras positivas de Mycoplasma gallisepticum de pollos en Ecuador.
| Isolates | Country | Year | GyrAa,c | ParCb,c | ||||
|---|---|---|---|---|---|---|---|---|
| 59 | 83 | 87 | 157 | 45 | 80 | |||
| S6 | USA | < 1968 | (His, H) CAT | (Ser, S) AGT | (Glu, E) GAA | Ile (I) ATT | Val (V) GTT | Ser (S) TCA |
| SYR-2 | Israel | 2015 | (His, H) | (Ser, S) | (Lys, K) | Val (V) | Ile (I) | Leu (L) |
| AVMG1 | Ecuador | 2019 | - | - | - | - | Val (V) | Trp (W) |
| AVMG2 | Ecuador | 2019 | (Tyr, Y) | (Ile, I) | (Glu, E) | Val (V) | Val (V) | Trp (W) |
| AVMG3 | Ecuador | 2019 | (His, H) | (Ile, I) | (Glu, E) | Val (V) | - | - |
| AVMG4 | Ecuador | 2019 | (His, H) | (Ile, I) | (Glu, E) | Val (V) | Val (V) | Trp (W) |
| AVMG5 | Ecuador | 2019 | (His, H) | (Ile, I) | (Glu, E) | Val (V) | - | - |
| AVMG6 | Ecuador | 2019 | (His, H) | (Ile, I) | (Glu, E) | Val (V) | Val (V) | Trp (W) |
The complete genome of Mycoplasma gallisepticum strain S6 (accession number CP006916) was used as reference strain, where amino acid positions indicated the relative position to the proteins:
a846 aa of GyrA DNA gyrase subunit A (Protein ID: AHB99967);
b796 aa of ParC DNA topoisomerase IV subunit A (Protein ID: AHB99685);
caa substitutions within QRDRs of Mycoplasma gallisepticum positive sample (i.e., AVMG1 to AVMG6) in comparison to the reference strain Mycoplasma gallisepticum strain S6 are marked.
Figure 1 Alignment of the quinolone resistance-determining region (QRDR) of gyrA gene of M. gallisepticum positive samples with an quinolone-sensitive strain of M. gallisepticum (strain S6). / Alineamiento de la región determinante de la resistencia a las quinolonas (QRDR) del gen gyrA de las muestras positivas de M. gallisepticum con una cepa de M. gallisepticum sensible a las quinolonas (cepa S6).
Figure 2 Alignment of the quinolone resistance-determining region (QRDR) of parC gene of M. gallisepticum positive samples with those of other quinolone-sensitive strain of M. gallisepticum (strain S6) and quinolone-resistance strains of M. gallisepticum (MDE-3, MYZ-8, YDK-4, and SyR-2). / Alineamiento de la región determinante de la resistencia a las quinolonas (QRDR) del gen parCde las muestras positivas de M. gallisepticum con las de otras cepas de M. gallisepticum sensibles a las quinolonas (cepa S6) y cepas de M. gallisepticum resistentes a las quinolonas (MDE-3, MYZ-8, YDK-4 y SyR-2).
