Juan Tatay-Dualde

In vitro assessment of the antimicrobial susceptibility of caprine isolates of Mycoplasma mycoides subsp. capri

The minimum inhibitory concentration (MIC) and minimum mycoplasmacidal concentration (MMC) of 17 antimicrobials against 41 Spanish caprine isolates of Mycoplasma mycoides subsp. capri (Mmc) obtained from different specimens (milk, external auricular canal and semen) were determined using a liquid microdilution method. For half of the isolates, the MIC was also estimated for seven of the antimicrobials using an epsilometric test (ET), in order to compare both methods and assess the validity of ET. Mutations in genes gyrA, gyrB, parC and parE conferring fluoroquinolone resistance, which have been recently described in Mmc, were investigated using PCR. The anatomical origin of the isolate had no effect on its antimicrobial susceptibility. Moxifloxacin and doxycycline had the lowest MIC values. The rest of the fluoroquinolones studied (except norfloxacin), together with tylosin and clindamycin, also had low MIC values, although the MMC obtained for clindamycin was higher than for the other antimicrobials. For all the aminoglycosides, spiramycin and erythromycin, a notable level of resistance was observed. The ET was in close agreement with broth microdilution at low MICs, but not at intermediate or high MICs. The analysis of the genomic sequences revealed the presence of an amino acid substitution in codon 83 of the gene gyrA, which has not been described previously in Mmc.

Molecular resistance mechanisms of Mycoplasma agalactiae to macrolides and lincomycin

The extensive use of antimicrobials for disease control has caused a remarkable decrease in antimicrobial susceptibility of different animal mycoplasma species, including Mycoplasma agalactiae (M. agalactiae), the main causative agent of contagious agalactia. However, the molecular mechanisms behind M. agalactiae resistance to macrolides and lincomycin have not yet been elucidated. The aim of the present study was to investigate the association between minimum inhibitory concentration (MIC) values of different antimicrobials and mutations in the 23S rRNA gene and ribosomal proteins L4 and L22, analysing both field isolates (n=50) and in vitro selected resistant mutants of M. agalactiae. The obtained MIC results of the studied field isolates demonstrate an increasing development of tylosin resistance in this bacterium, in comparison to previous studies. Interestingly, predicted amino acid changes in L22 (Ser89Leu and Gln90Lys/His) were the first variations observed when MICs of M. agalactiae started to increase (tylosin MIC ≥0.8μg/ml), whereas mutations at positions 2058 or 2059 of domain V of the 23S rRNA gene appeared from MIC values of 1.6μg/ml. These results were consistent in both field isolates and in vitro selected mutants of M. agalactiae. Thus, although in other mycoplasma species resistance to macrolides and lincosamides had been mainly related to mutations in the 23S rRNA gene, this work demonstrates the role of alterations in ribosomal protein L22 in decreased susceptibility of M. agalactiae. Moreover, these mutations can be used as molecular markers to set an interpretative breakpoint of antimicrobial resistance for M. agalactiae.

Mutations in the quinolone resistance determining region conferring resistance to fluoroquinolones in Mycoplasma agalactiae

M. agalactiae is the main causative agent of contagious agalactia, against which antimicrobial treatment is the main applied control measure. Quinolones are an effective group of antimicrobials inhibiting the growth of M. agalactiae, but in the last years, various reports have demonstrated an increase of resistance in field isolates due to its massive use. Nevertheless, the molecular mechanisms involved in the acquisition of fluoroquinolones resistance in M. agalactiae have not been elucidated yet. Therefore, the aim of this work was to analyze the presence of DNA variations that could be related to changes in fluoroquinolone susceptibility. For this purpose, three M. agalactiae strains were selected to obtain in vitro resistant mutants against enrofloxacin, marbofloxacin and moxifloxacin and afterwards, partial sequences of their gyrA, gyrB, parC and parE genes were analyzed. In addition, a set of field isolates with different MIC values were also studied. Changes related to variations in fluoroquinolones susceptibility were found in gyrB, parC and parE. Specifically, gyrB genes were affected at the predicted amino acid position 424, four amino acid changes were detected in parC (positions 78, 79, 80 and 84) and two substitutions were reported in parE (amino acid positions 429 and 459). Mutations at predicted positions 424 of gyrB and 429 of parE are novel DNA changes which had not been previously described and, on the whole, parC was the first gene showing alterations when changes in susceptibility to fluoroquinolones occurred. Thus, this gene is the most suitable target for a rapid study of fluoroquinolone resistance in field isolates of M. agalactiae.

Antimicrobial susceptibility and multilocus sequence typing of Mycoplasma capricolum subsp. capricolum

Mycoplasma capricolum subsp. capricolum is one of the causative agents of contagious agalactia (CA). Nevertheless, there is still a lack of information about its antimicrobial susceptibility and genetic characteristics. Therefore, the aim of this work was to study the antimicrobial and genetic variability of different Mycoplasma capricolum subsp. capricolum field isolates. For this purpose, the growth inhibition effect of 18 antimicrobials and a multilocus sequence typing (MLST) scheme based on five housekeeping genes (fusA, glpQ, gyrB, lepA and rpoB) were performed on 32 selected field isolates from Italy and Spain.The results showed a wide range of growth inhibitory effects for almost all the antimicrobials studied. Macrolides presented lower efficacy inhibiting Mcc growth than in previous works performed on other CA-causative mycoplasmas. Erythromycin was not able to inhibit the growth of any of the studied strains, contrary to doxycycline, which inhibited the growth of all of them from low concentrations. On the other hand, the study of the concatenated genes revealed a high genetic variability among the different Mcc isolates. Hence, these genetic variations were greater than the ones reported in prior works on other mycoplasma species.

Multilocus sequence typing of Mycoplasma mycoides subsp. capri to assess its genetic variability in a contagious agalactia endemic area

Mycoplasma mycoides subsp. capri (Mmc) is one of the main causative agents of caprine contagious agalactia. Besides, the absence of accurate control methods eases its dispersion between different herds within endemic areas of this disease. In this context, there is a need to implement molecular typing schemes which offer valuable information useful to establish control measures and enables the surveillance of this pathogen. The aim of this study was to assess the genetic variability of different strains of Mmc from a contagious agalactia endemic area through multilocus sequence typing (MLST). For this purpose, five house-keeping genes (fusA, glpQ, gyrB, lepA, rpoB) from 39 field isolates were analysed. These isolates were obtained from different geographic areas of Spain, between the years 2004 and 2015. The results obtained in this study suggest that the selected MLST scheme could be a useful technique to monitor the genetic variability of Mmc in endemic areas. Despite the significant differences found between the assessed field isolates, they could be classified according to their geographical origin. Moreover, it was also possible to detect genetic differences between Mmc strains coming from the same herd at the same sampling time, which may need to be taken into consideration when designing or arranging prophylactic strategies.

Presence of Mycoplasma agalactiae in semen of naturally infected asymptomatic rams.

The purpose of the present study was to assess the presence of Mycoplasma agalactiae (Ma), the main causative agent of ovine contagious agalactia (CA), in semen of naturally infected rams. Therefore, semen samples from 167 rams residing in three different artificial insemination (AI) centers of a CA-endemic area were studied by microbiological and molecular techniques. In addition, serial ejaculates from the same rams were evaluated to determine the excretion dynamics of Ma. Of the 384 samples studied, Ma was detected in 56 (14.58%) which belonged to 44 different rams (26.35%). These findings confirm the ability of Ma to be excreted in semen of asymptomatic rams. Furthermore, these results also evidence the presence of these asymptomatic carriers of Ma in ovine AI centers, representing a serious health risk regarding the spread and maintenance of CA, especially in endemic areas. Moreover, the excretion of Ma in semen also points to the risk of venereal transmission of this disease. The current results highlight the need to implement control measures to prevent the admission of infected rams in AI centers and the necessity to continuously monitor semen samples to effectively detect infected individuals.

23S rRNA and L22 ribosomal protein are involved in the acquisition of macrolide and lincosamide resistance in Mycoplasma capricolum subsp. capricolum

Mycoplasma capricolum subsp. capricolum (Mcc) is one of the causative agents of contagious agalactia, and antimicrobial therapy is the most commonly applied measure to treat outbreaks of this disease. Macrolides and lincosamides bind specifically to nucleotides at domains II and V of the 23S rRNA. Furthermore, rplD and rplV genes encode ribosomal proteins L4 and L22, which are also implicated in the macrolide binding site. The aim of this work was to study the relationship between mutations in these genes and the acquisition of macrolide and lincosamide resistance in Mcc. For this purpose, in vitro selected resistant mutants and field isolates were studied. This study demonstrates the appearance of DNA point mutations at the 23S rRNA encoding genes (A2058G, A2059G and A2062C) and rplV gene (Ala89Asp) in association to high minimum inhibitory concentration values. Hence, it proves the importance of alterations in 23S rRNA domain V and ribosomal protein L22 as molecular mechanisms responsible for the acquisition of macrolide and lincosamide resistance in both field isolates and in vitro selected mutants. Moreover, these mutations enable us to provide an interpretative breakpoint of antimicrobial resistance for Mcc at MIC 0.8 μg/ml.

The moderate drift towards less tetracycline-susceptible isolates of contagious agalactia causative agents might result from different molecular mechanisms

Contagious agalactia is a mycoplasmosis that affects small ruminants, is associated with loss of milk production and high morbidity rates, and is highly deleterious to dairy industries. The etiological agents are four mycoplasma (sub)species, of which the relative importance depends on the countries and the animal host. Tetracyclines are non-expensive, broad-spectrum antimicrobials and are often used to control mastitis in dairy herds. However, the in vitro efficiency of tetracyclines against each of the etiological agents of contagious agalactia has been poorly assessed. The aims of this study were i) to compare the tetracycline susceptibilities of various field isolates, belonging to different mycoplasma (sub)species and subtypes, collected over the years from different clinical contexts in France or Spain, and ii) to investigate the molecular mechanisms behind the decreased susceptibility of some isolates to tetracyclines. The Minimum Inhibitory Concentrations (MICs) of tetracyclines were determined in vitro on a set of 120 isolates. Statistical analyses were run to define the significance of any observed differences in MICs distribution. As mutations in the genes encoding the tetracycline targets (rrs loci) are most often associated with increased tetracycline MICs in animal mycoplasmas, these genes were sequenced. The loss of susceptibility to tetracyclines after year 2010 is not significant and recent MICs are higher in M. agalactiae, especially isolates from mastitis cases, than in other etiological agents of contagious agalactia. The observed increases in MICs were not always associated with mutations in the rrs alleles which suggests the existence of other resistance mechanisms yet to be deciphered.

Resistance mechanisms against quinolones in Mycoplasma capricolum subsp. capricolum

Quinolones interact with bacterial DNA gyrase and topoisomerase IV, the subunits of which are encoded by gyrA/gyrB and parC/parE, respectively. The aim of this study was to evaluate the relationship between changes in these genes and quinolone susceptibility of Mycoplasma capricolum subsp. capricolum (Mcc). Using in vitro selected resistant mutants and field isolates from goats, predicted amino acid changes in gyrA, gyrB and parC were associated with higher minimum inhibitory concentration values for quinolones. Alterations in parC predicted amino acid sequences were most frequently associated with quinolone resistance in Mcc.

Detecting asymptomatic rams infected with Mycoplasma agalactiae in ovine artificial insemination centers

Mycoplasma agalactiae (Ma) is the main causative agent of ovine contagious agalactia, which is a serious disease of small ruminants. In endemic areas, its most common clinical situation consists of chronically infected herds, and asymptomatic infected individuals represent an epidemiological risk regarding the transmission of this disease. The aim of this work was to detect the presence of asymptomatic rams infected with Ma in different artificial insemination centers, and to determine the most effective way to identify these individuals so as to implement adequate surveillance protocols. For this purpose, 215 rams and 14 teaser sheep were sampled taking auricular, nasal, and vaginal swabs and serum samples. In addition, ejaculates from 147 rams were analyzed. These samples were subjected to specific culture and molecular techniques to isolate and identify mycoplasmas, and to a serological test to detect antibodies against Ma. Mycoplasma agalactiae was detected in 47 (4.4%) of the 1077 samples analyzed, and also one individual resulted seropositive. Thus, 37 (17.2%) of the 215 studied rams were infected with Ma. The specimens which proportionally yielded the greatest number of positive results for this pathogen were semen samples (13.6%), followed by nasal swabs (5.8%). In contrast, the sampling of the external auricular canal and the serological analyses resulted insufficient to effectively detect infected individuals. Asymptomatic rams infected with Ma were detected in all the analyzed artificial insemination centers, highlighting the need to implement adequate surveillance protocols to prevent the presence of these individuals in these centers, reducing the risk of transmitting contagious agalactia.