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

© 2018. Elsevier. This document is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Accepted version of a Published Work that appeared in final form in Veterinary Microbiology. To access the final edited and published work see https://doi.org/10.1016/j.vetmic.2018.02.014Mycoplasma capricolum subsp. capricolum (Mcc) is one of the causative agents of contagious agalactia, and antimicrobial treatment 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 gene. 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 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 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. Furthermore, these mutations enable us to provide an interpretative breakpoint of antimicrobial resistance for Mcc at MIC 0.8 µg/ml

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

Tipificación molecular y mecanismos de resistencia antimicrobiana en micoplasmas asociados a la agalaxia contagiosa

La presente tesis doctoral se ha realizado en formato de compendio de publicaciones y los artículos que la conforman tienen el objetivo común de evaluar distintas técnicas disponibles para la detección de M. agalactiae en muestras de leche de cabra, así como la tipificación molecular de las cepas circulantes y los mecanismos de resistencia antimicrobiana de las micoplasmas causantes de la agalaxia contagiosa. De esta forma, los objetivos específicos y la metodología seguida fueron: 1) Evaluar los límites de detección de las técnicas de cultivo microbiológico y PCR empleadas para la detección de M. agalactiae en muestras de leche de cabra inoculadas experimentalmente; 2) Determinar la concordancia entre las técnicas de cultivo microbiológico y PCR utilizadas para detectar M. agalactiae en muestras de mamitis procedentes de rebaños caprinos crónicamente infectados; 3) Evaluar la variabilidad genética de cepas de campo de M. mycoides subsp. capri y M. capricolum subsp. capricolum con diferentes características epidemiológicas procedentes de áreas endémicas de agalaxia contagiosa; 4) Evaluar la susceptibilidad in vitro a diferentes antimicrobianos de cepas de M. capricolum subsp. capricolum aisladas en España e Italia y 5) Estudiar la importancia de los genes de la región QRDR en el aumento de la resistencia a quinolonas así como determinar las mutaciones específicas que nos aportan información acerca de la susceptibilidad a quinolonas de las cepas de M. agalactiae y M. capricolum subsp. capricolum. Los resultados obtenidos permiten concluir que: 1) En muestras de leche de cabra, la técnica del cultivo microbiológico presenta un límite de detección de M. agalactiae inferior al obtenido por la PCR. Por lo tanto, en muestras de leche de tanque se recomienda la realización del diagnóstico mediante cultivo, mientras que en muestras de mamitis clínicas ambas técnicas presentan una buena concordancia; 2) Los esquemas de MLST son una herramienta útil para la detección de diferencias genéticas entre aislamientos de M. mycoides subsp. capri procedentes de distintos orígenes geográficos. Asimismo, esta técnica permite detectar diferencias entre cepas presentes en un mismo rebaño al mismo tiempo; 3) M. mycoides subsp. capri y M. capricolum subsp. capricolum presentan una gran variabilidad genética entre cepas aisladas en una misma área endémica de agalaxia contagiosa, mayor que la observada anteriormente en otras especies de micoplasmas como M. agalactiae; 4) No existe una relación directa entre la evolución filogenética de las cepas de M. capricolum subsp. capricolum y sus perfiles de susceptibilidad a distintos antimicrobianos; 5) La doxiciclina es el antimicrobiano más eficaz para inhibir el crecimiento de las cepas estudiadas de M. capricolum subsp. capricolum. Asimismo, algunos de los aislamientos analizados resultaron ser resistentes a quinolonas y macrólidos; 6) El aumento de la resistencia a quinolonas en M. capricolum subsp. capricolum y M. agalactiae se debe a la aparición de mutaciones en los genes que codifican la DNA girasa y la topoisomerasa IV bacterianas y 7) El gen parC es el que aporta mayor información acerca de la susceptibilidad a quinolonas de M. capricolum subsp. capricolum y M. agalactiae.This doctoral thesis is presented as a compendium of publications and the articles that form part of it have the common goal of evaluate different diagnostic techniques for the detection of M. agalactiae in goat milk samples and the characterization of field isolates of contagious agalactia causing mycoplasmas by molecular typing and the study of their antimicrobial resistance mechanisms. Thus, the specific objectives and methods were: 1) To determine the lower M. agalactiae detection limits of culture and PCR techniques in experimentally inoculated goat milk samples, 2) To establish the agreement between PCR and culture techniques for the detection of M. agalactiae using mastitic milk samples retrieved from chronically infected commercial dairy herds, 3) To assess the genetic variability of different M. mycoides subsp. capri and M. capricolum subsp. capricolum field isolates with different epidemiological characteristics isolated from contagious agalactia endemic areas, 4) To evaluate the in vitro antimicrobial susceptibility of M. capricolum subsp. capricolum field isolates retrieved from Spain and Italy and 5) To study the importance of the QRDR in the acquisition of quinolone resistance, determining the specific genes that provide useful information about quinolone resistance in M. agalactiae and M. capricolum subsp. capricolum isolates. The obtained results allowed us to conclude: 1) In goat milk samples, bacterial culture technique has a lower detection limit of M. agalactiae than PCR. Therefore, culture is recommended when bulk tank milk samples are analyzed, whereas both techniques have a good agreement when detecting this pathogen in mastitic milk samples; 2) MLST schemes are a useful tool to detect genetic differences between different M. mycoides subsp. capri field isolates coming from different geographical areas. Moreover, they also allow the detection of variations between isolates retrieved from the same herd at the same time; 3) M. mycoides subsp. capri and M. capricolum subsp. capricolum show great genetic variability between field isolates coming from the same contagious agalactia endemic area. These differences are greater than those reported in other mycoplasma species such as M. agalactiae; 4) There is no connection between phylogeny and antimicrobial susceptibility in the studied M. capricolum subsp. capricolum strains; 5) Doxycycline is the most effective antimicrobial inhibiting M. capricolum subsp. capricolum growth. Besides, some of the analyzed field isolates were resistant to quinolones and macrolides; 6) The acquisition of quinolone resistance in M. capricolum subsp. capricolum and M. agalactiae is due to mutations in the genes that encode enzymes DNA gyrase and topoisomerase IV and 7) parC is the gene providing most information about quinolone susceptibility in M. capricolum subsp. capricolum and M. agalactiae

Mycoplasma excretion in reproductive male and female goats

Three experiments were designed in order to study the excretion routes of mycoplasmas involved in caprine contagious agalactia in chronically infected goat populations. The results obtained for external auricular canal (EAC) swabs were compared to other samples: 1) nasal swabs in 95 bucks; 2) conjunctival swabs in 85 bucks; 3) milk and vaginal swabs in 23 goats. Serology to detect specific antibodies against Mycoplasma agalactiae (Ma) was also conducted. Out of 1061 samples, the presence of Ma, Mmc and Mcc was detected in 23, 18 and 8 samples, respectively, which belonged to 24 bucks and 7 goats. EAC analyses detected 21 bucks (87.5%) and 4 infected goats (57.1%). Additional sampling (conjunctival, nasal swabs and vaginal swabs in goats) allowed the detection of further infected animals, in comparison to the sole sampling of the EAC

Zoonosis y salud laboral en la profesión veterinaria

ABSTRACT 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.RESUMEN 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

Zoonoses in Veterinary Students: A Systematic Review of the Literature

<div><p>Background</p><p>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.</p><p>Objectives</p><p>The present study utilizes a systematic review of the literature to identify zoonoses described in veterinary students.</p><p>Data sources</p><p>Web of Science and PubMed.</p><p>Results</p><p>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.</p><p>Limitations</p><p>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.</p><p>Conclusions/implications</p><p>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.</p></div

Zoonoses reported in veterinary students identified by the systematic literature review according to the agent, country, year and main quantitative and qualitative information summarized from the selected studies.

<p>Zoonoses reported in veterinary students identified by the systematic literature review according to the agent, country, year and main quantitative and qualitative information summarized from the selected studies.</p

Flowchart of the selection process for publications selected in this review.

<p>Flowchart of the selection process for publications selected in this review.</p