Efflux Might Participate in Decreased Susceptibility to Oxytetracycline in Contagious Agalactia-Causative Mycoplasma spp.

Contagious agalactia is associated with mastitis, keratoconjunctivitis, arthritis, pneumonia, and septicemia in small ruminants in countries with large dairy industries worldwide. The causative agents belong to four (sub)species of the Mycoplasma genus that have remained essentially susceptible to antimicrobials, including to the widely-used tetracycline family. However, some clinical isolates have been detected that show increased minimum inhibitory concentrations of tetracyclines, although they do not harbor the mutation in the 16SrRNA gene usually associated with resistance. The present work aimed to assess whether efflux pumps, infrequently described in mycoplasmas, could participate in the observed moderate loss of susceptibility. General efflux mechanisms were measured (i) using the fluorescence property of ethidium bromide when accumulated intracellularly and intercalated in the mycoplasma genomes, its active extrusion resulting in a temperature-dependent decrease in fluorescence and (ii) monitoring the growth inhibition of mycoplasmas by subinhibitory concentrations of tetracycline with or without reserpine, a known inhibitor of efflux in other bacteria. Both methods revealed non-specific efflux phenomena in most of the isolates tested, although their efficacy was difficult to quantify. This property could contribute to the acquisition of mutations conferring resistance by maintaining intracellular concentrations of tetracyclines at subinhibitory levels.

[Zoonoses and occupational health in the veterinary profession]. / Zoonosis y salud laboral en la profesión veterinaria.

The veterinary profession implies a greater risk of infection by zoonotic pathogens than the overall population. The aim of the present work was to evaluate the impact of zoonoses on the occupational health of veterinarians reviewing the published surveys addressing this subject. Following these inquiries, between 4% and 64.3% of the surveyed professionals acknowledge to have suffered at least one zoonotic disease, and dermatophytosis is the most frequently described zoonosis. In addition, from a qualitative point of view, it is necessary to highlight the occurrence of different diseases of important clinical seriousness and the existence of infections by antimicrobial-resistant pathogens. Due to the absence of updated studies in Spain, the present work reflects on the need to recognize the health determinants associated to the veterinary profession within the characteristics of our labour market. The available data suggest that the occurrence of zoonoses as occupational diseases is underestimated in Spain. Therefore, performing surveys on occupational health, documenting and publishing cases and reviewing the risks and the impact of zoonotic diseases on the veterinary profession would contribute to the description and notification of such diseases on behalf of the healthcare administration and would also become an essential tool in occupational risk prevention.

La profesión veterinaria presenta mayor riesgo de infección frente a agentes patógenos de carácter zoonósico que la población general. Con el objetivo de valorar el impacto de las zoonosis, el presente trabajo revisa las encuestas publicadas sobre salud laboral en la profesión veterinaria. En dichas encuestas, el rango de profesionales que refieren haber padecido al menos una zoonosis oscila entre el 4% y el 64,3%, siendo la dermatofitosis la zoonosis comunicada con mayor frecuencia. Además, desde el punto de vista cualitativo, cabe destacar la comunicación de diferentes procesos de especial gravedad clínica, así como la existencia documentada de infecciones por agentes patógenos resistentes a los antimicrobianos. Dada la ausencia de estudios actualizados en España, se reflexiona sobre la necesidad de conocer los determinantes de salud asociados a las características del mercado laboral de la profesión en nuestro entorno. Los datos existentes sugieren que la consideración legal de enfermedad profesional para las zoonosis se encuentra infraestimada en nuestro país. Por todo ello, la realización de encuestas de salud laboral, la documentación y publicación de los casos, así como la revisión de los riesgos y el impacto de las zoonosis en la profesión veterinaria, pueden contribuir a la calificación y notificación de dichas enfermedades por parte de la administración sanitaria, al tiempo que suponen una herramienta fundamental en el ámbito de la prevención de riesgos laborales.

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.

Zoonosis y salud laboral en la profesión veterinaria / Zoonoses and occupational health in the veterinary profession

La profesión veterinaria presenta mayor riesgo de infección frente a agentes patógenos de carácter zoonósico que la población general. Con el objetivo de valorar el impacto de las zoonosis, el presente trabajo revisa las encuestas publicadas sobre salud laboral en la profesión veterinaria. En dichas encuestas, el rango de profesionales que refieren haber padecido al menos una zoonosis oscila entre el 4% y el 64,3%, siendo la dermatofitosis la zoonosis comunicada con mayor frecuencia. Además, desde el punto de vista cualitativo, cabe destacar la comunicación de diferentes procesos de especial gravedad clínica, así como la existencia documentada de infecciones por agentes patógenos resistentes a los antimicrobianos. Dada la ausencia de estudios actualizados en España, se reflexiona sobre la necesidad de conocer los determinantes de salud asociados a las características del mercado laboral de la profesión en nuestro entorno. Los datos existentes sugieren que la consideración legal de enfermedad profesional para las zoonosis se encuentra infraestimada en nuestro país. Por todo ello, la realización de encuestas de salud laboral, la documentación y publicación de los casos, así como la revisión de los riesgos y el impacto de las zoonosis en la profesión veterinaria, pueden contribuir a la calificación y notificación de dichas enfermedades por parte de la administración sanitaria, al tiempo que suponen una herramienta fundamental en el ámbito de la prevención de riesgos laborales

The veterinary profession implies a greater risk of infection by zoonotic pathogens than the overall population. The aim of the present work was to evaluate the impact of zoonoses on the occupational health of veterinarians reviewing the published surveys addressing this subject. Following these inquiries, between 4% and 64.3% of the surveyed professionals acknowledge to have suffered at least one zoonotic disease, and dermatophytosis is the most frequently described zoonosis. In addition, from a qualitative point of view, it is necessary to highlight the occurrence of different diseases of important clinical seriousness and the existence of infections by antimicrobial-resistant pathogens. Due to the absence of updated studies in Spain, the present work reflects on the need to recognize the health determinants associated to the veterinary profession within the characteristics of our labour market. The available data suggest that the occurrence of zoonoses as occupational diseases is underestimated in Spain. Therefore, performing surveys on occupational health, documenting and publishing cases and reviewing the risks and the impact of zoonotic diseases on the veterinary profession would contribute to the description and notification of such diseases on behalf of the healthcare administration and would also become an essential tool in occupational risk prevention

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.

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.

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.

Zoonoses in Veterinary Students: A Systematic Review of the Literature.

BACKGROUND: Veterinary students face diverse potential sources of zoonotic pathogens since the first years of their academic degree. Such sources include different animal species and pathologic materials which are used at university facilities as well as commercial clinics, farms and other external facilities. OBJECTIVES: The present study utilizes a systematic review of the literature to identify zoonoses described in veterinary students. DATA SOURCES: Web of Science and PubMed. RESULTS: Of the 1,254 titles produced by the bibliographic search, 62 were included in this review. Whereas 28 of these articles (45.2%) described individual cases or outbreaks, the remaining 34 (54.8%) reported serological results. The zoonotic etiological agents described were bacteria, in 39 studies (62.9%), parasites, in 12 works (19.4%), virus, in 9 studies (14.5%) and fungi, in 2 (3.2%) of the selected articles. The selected literature included references from 24 different countries and covered the time period of the last 55 years. LIMITATIONS: The fact that common cases of disease or cases of little clinical importance without collective repercussions are not usually published in peer-reviewed journals limits the possibility to reach conclusions from a quantitative point of view. Furthermore, most of the selected works (66.1%) refer to European or North American countries, and thus, the number of cases due to pathogens which could appear more frequently in non-occidental countries might be underestimated. CONCLUSIONS/IMPLICATIONS: The results of the present systematic review highlight the need of including training in zoonotic diseases since the first years of Veterinary Science degrees, especially focusing on biosecurity measures (hygienic measures and the utilization of the personal protective equipment), as a way of protecting students, and on monitoring programs, so as to adequately advise affected students or students suspicious of enduring zoonoses.

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.

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.