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Fate of non O157 Shigatoxigenic Escherichia coli in ovine manurecomposting

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http://dx.doi.org/10.1590/1678-6001 Arq. Bras. Med. Vet. Zootec., v.66, n.6, p.1771-1778, 2014 Fate of non O157 Shigatoxigenic Escherichia coli in ovine manure composting [Eliminação de Escherichia coli Shigatoxigênica não O157 em compostagem de esterco ovino] J.M. Marin1, R.P. Maluta2, C.A. Borges2, L.G. Beraldo2, S.A. Maesta3, M.V.F. Lemos4, U.S. Ruiz3, F.A. Ávila4, E.C. Rigobelo3 1Universidade de São Paulo − FORP − Ribeirão Preto, SP 2Aluno de pós-graduação − FCAV − Universidade Estadual Paulista-Unesp − Jaboticabal, SP 3Unesp − Campus de Dracena − Dracena, SP 4FCAV-Unesp − Jabotricabal, SP ABSTRACT Livestock manure may contain pathogenic microorganisms which pose a risk to the health of animal or humans if the manure is not adequately treated or disposed of. To determine the fate of Shiga toxigenic Escherichia coli (STEC) non O157 in composted manure from naturally colonized sheep, fresh manure was obtained from animals carrying bacterial cells with stx1/ stx2 genes. Two composting systems were used, aerated and non-aerated, and the experiments were done in Dracena city, São Paulo State. Every week, for seven weeks, one manure sample from six different points in both systems was collected and cultured to determine the presence of E. coli, the presence of the virulence genes in the cells, and also the susceptibility to 10 antimicrobial drugs. The temperature was verified at each sampling. STEC non-O157 survived for 49 days in both composting systems. E. coli non-STEC showing a high degree of antibiotic resistance was recovered all long the composting period. No relationship was established between the presence of virulence genes and antibiotic resistance. The presence of virulence genes and multiple antibiotic resistances in E. coli implicates a potential risk for these genes spread in the human food chain, which is a reason for concern. Keywords: bacterial elimination, VTEC, environment, compost, virulence gene RESUMO Esterco de animais de criação pode conter microrganismos patogênicos, o que representa um risco para a saúde animal e a humana se o esterco não for adequadamente tratado ou descartado. Determinou-se o tempo necessário para a eliminação de Escherichia coli Shiga toxigenica (STEC) não O157 em esterco ovino composto, obtido de fezes frescas de ovelhas naturalmente colonizadas com cepas STEC não O157 que apresentavam os genes stx1/ stx2. Foram utilizados dois sistemas de compostagem, aerado e não aerado, em experimentos realizados na cidade de Dracena, estado de São Paulo. Todas as semanas, durante sete semanas, uma amostra de compostagem proveniente de seis pontos diferentes na leira, nos dois sistemas, foi coletada e semeada para a determinação da presença de E. coli, da presença de genes de virulência nas células, bem como da sensibilidade dessas células a 10 drogas antimicrobianas. Em cada amostragem, a temperatura da leira foi analisada. Células de STEC não O157 sobreviveram por 49 dias nos dois sistemas de compostagem. E. coli não STEC com um alto grau de resistência a antibióticos foi recuperada ao longo de todo o período de compostagem. Não foi possível estabelecer relação entre a presença de genes de virulência e a resistência a antibióticos. A presença de genes de virulência e a resistência a múltiplos antibióticos em E. coli representam um risco potencial para o espalhamento desses genes na cadeia alimentar humana, o que é motivo de grande preocupação. Palavras-chave: eliminação de bactéria, VTEC, meio ambiente, esterco, genes de virulência Recebido em 25 de julho de 2012 Aceito em 4 de agosto de 2014 E-mail: jmmarin@forp.usp.br Marin et al. INTRODUCTION Composting is a traditional way to treat livestock manure to make it easier to dispose of on land, and to produce an inexpensive fertilizer. However, livestock manure may contain zoonotic microbial pathogens, including Salmonella spp and Shiga toxigenic Escherichia coli (STEC) producing a Shiga-like toxin (stx1 and/or stx2) (Nicholson et al., 2005). Since the emergence of STEC as human pathogens, contamination of foods of animal origin has been a major public health concern. Ruminants, among them cattle and sheep, sporadically harbor STEC strains in their gastrointestinal tracts and shed the bacteria in their feces (Kudva et al., 1998). E. coli O157:H7 is one of the leading causes of bacterial diarrhea and hemorrhagic colitis, showing two antigenically different forms of Shiga toxin, the stx1 and stx2. The key virulence factor for STEC is Stx that cleaves ribosomal RNA, causing protein synthesis to cease. The Stx family contains two subgroups Stx1 and Stx2 that share approximately 55% amino acid homology. In the kidney the Stx damages the renal endothelium cells and occludes the microvasculature resulting in renal inflammation. Stx also induces apoptosis in intestinal epithelial cells and local damage to the colon which results in blood diarrhea, hemorrhagic colitis, necrosis and intestinal perforation (Kaper et al., 2004). It has emerged as a foodborne pathogen and has been implicated in over 20,000 cases of infection and 250 deaths per year in the United States (Koutkia et al., 1997). Outbreaks have been traced to ground beef, raw milk, water, fruits and vegetables that have been contaminated with this pathogen (Lung et al., 2001). While serotype O157:H7 is the best-recognized strain, other serotypes are responsible for approximately half of all confirmed STEC infections in Europe (Bolton et al., 2011), and there is a growing concern about the risk of non-O157:H7 STEC serotypes to human health (Fremaux et al., 2007; Bolton et al., 2011). Transfer of STEC strains to humans through contact with contaminated environments is becoming increasingly recognized as a major route of human infection, particularly infection among children (Strachan et al., 2006). A number of previous studies have shown that E. coli can survive for up to several months in experimentally inoculated animal feces (Kudva et al., 1998; Lung et al., 2001; Asano et al., 2011). The disinfection of manure during composting is based on the time-temperature relationship that kills the pathogens plus the action of antagonistic microorganisms during this process (Himathongkham and Rieman, 1999; Turner, 2002; Larney et al., 2003). The E. coli strains encountered in manure can present a high degree of antimicrobial resistance. This resistance to antimicrobials is the result of the dissemination of genetic elements such as plasmids, transposons and integrons. However, there are few data regarding antimicrobial resistance to the virulence genes of STEC (Zhang et al., 2000; Mora et al., 2005) and previous studies on this issue are controversial (Clermont et al., 2008). The use of antibiotics during animal production has been a controversial subject due its potential to transfer antimicrobial resistance genes to both animals and humans. Nagachinta et al. (2009) suggested a possible relationship between conjugative plasmid in the dissemination of antimicrobial resistance genes together with pathogenicity genes. There is limited information as to whether the presence of antimicrobial resistance genes in E. coli strains increases their survival times during the composting process. In the present study, the aims were to determine the fate of STEC nonO157 cells in ovine manure in two different composting systems as well as to verify if there is a relationship between antimicrobial resistance patterns and virulence factors. MATERIAL AND METHODS During March 2010 anal swabs were collected from the rectum of ovines in a herd of 65 healthy adult sheep from a facility owned by the Graduate School of Agriculture located in Dracena city in the Northwestern region of São Paulo State, Brazil. The swabs were transferred to a bottle containing Stuart Transport Medium (BBL/Becton Dickinson, Sparks, MD, USA) and were aerobically cultured in MacConkey agar (Oxoid, Basingstoke, UK), as soon as possible. E. coli isolates were identified based on the colony characteristics, Gram staining and biochemical profile (Farmer, 1999). At least five 1772 Arq. Bras. Med. Vet. Zootec., v.66, n.6, p.1771-1778, 2014 Fate of non O157 colonies per plate were selected for further analysis. The isolates were serotyped for serotype O157:H7 using the Latex Agglutination test Kit (Oxoid, Basingstoke, UK). Negative strains were considered non-O157. E. coli EDL 933 strain was used as a positive control for O157:H7 serotype. A loopfull of cells from each five colonies above selected was collected, mixed and grown overnight in Luria Bertani broth (Difco, Oxford, UK), at 37oC. Bacteria were pelleted from 1.5mL of broth, suspended in 200uL sterile distilled water and boiled for 10 min. Following centrifugation of the lysate, a 150uL sample of the supernatant was stored at -20oC as a template DNA stock (Keskimaki et al., 2001). Specific primers were used to amplify sequences of stx1, stx2 and eae genes. Primer sequences, predicted sizes of the amplified products, and the specific conditions were carried out according to those described by Vidal et al. (2005). Strains carrying stx1 and/or stx2 genes detected by PCR method were classified as a STEC strain and those without these genes were classified as a nonSTEC strain. The composting was performed at a field-scale facility owned by the School using fresh manure obtained from all sheep including those that have been confirmed to have non-O157 STEC strains possessing both, stx1 and stx2 genes. Five hundred kilograms of sheep feces were heaped for two treatments. The first one (A - aerated compost) was turned every three days during the 7 week treatment period (March to April 2010) while the second (B - non-aerated compost) was not turned and remained without revolving throughout the experiment. Three parts of dry grass and one part of sheep feces were added to each treatment to equilibrate the nutritional balance among nitrogen and carbon, which facilitates the decomposition of all material. The two heaps were 2m long by 2m wide by 0.5m deep. Water was added daily to maintain the saturation point at 60 %. Under the heaps, six equidistant points were traced, and samples were obtained weekly, on Monday. At each sampling time, the temperature of the compost was determined at each specified location before any internal samples were removed from the heaps. Once these data were collected, the compost heaps were opened with a shovel and the samples were collected aseptically. Twenty-five grams of compost samples were mixed with 225mL of Tryptone Soya Broth (Oxoid, Basingstoke, UK). Decimal dilution of each homogenate were prepared in maximum recovery diluents (MRD, Oxoid, Basingstoke, UK). E. coli were enumerated by spread-plating a volume of 100μL onto MacConkey agar (Oxoid, Basingstoke, UK) plates which were incubated at 37oC for 18 hours. Colonies were counted and titres obtained in a colony forming unit (CFU/mL). Ten colonies per plate were selected for E coli confirmation as described above. After that a loopfull of each colony was inoculated and grown overnight into Luria Bertani broth (Difco, Oxoid, UK). The template DNA stock was obtained as described above. The presence of bacteria containing the virulence genes was assayed by PCR as mentioned. The ten isolates mentioned above were examined for susceptibility to 10 antimicrobial drugs using the Bauer-Kirby disc diffusion method using commercial disks (CEFAR, São Paulo, Brazil). The following discs were used: ampicillin (Amp, 10μg), tetracycline (Tet, 30μg), trimethoprim (Tri, 25μg), erythromycin (Eri, 15μg), nalidixic acid (Nal, 30μg), streptomycin (Str, 30μg), novobiocin (Nov, 30μg), lincomycin (Lin, 10μg), penicillin (Pen, 10μg) and neomycin (Neo, 30μg). E. coli strain ATCC 25922 and Staphilococus aureus strain ATCC 29213 were used for quality control. Isolates were classed as sensitive, intermediate and resistant to each drug according to Committee for Laboratory Standards Institute (CLSI, 2008). RESULTS The average changes in E. coli number in the composting system are shown in Table 1. Samples were taken from six equidistant points inside the heap for both A and B composting treatments. Eighty-four compost samples were collected for further analysis from both composting (A=42; B=42), the temperature fluctuations were also analyzed during this time. For treatment A the suitable temperature for the reduction or elimination of the pathogenic microorganisms was reached in the first week around 55oC, while for B this marker was found only in the second week. A total of 695 E. coli isolates (321 from treatment A and 374 from treatment B) were obtained from the MacConkey plates during the seven weeks of experiment. Arq. Bras. Med. Vet. Zootec., v.66, n.6, p.1771-1778, 2014 1773 Marin et al. Although the total number of E. coli isolates carrying virulence genes decreased weekly throughout the experiment, the values found for treatment B (non-aerated) were higher than for treatment A (Figure 1A, B). Tab.2 shows all the isolates encoding virulence genes recovered in both treatments. The values for treatment B were 55, 65 and 30 and for treatment A 22, 49 and 6 respectively, for stx1, stx 2 and eae genes (Table 2). A decrease in the volume of material was larger for treatment A when compared with treatment B (data not shown) which was probably the result of a better decomposition. The serotype O157:H7 was not found among the isolates recovered from both treatments. The STEC strains carrying the stx2 gene (the most numerous virulence gene) and also non-STEC strains showed a high degree of antimicrobial resistance for the 10 drugs tested, as shown in Figure 2; there was a high similarity among the antimicrobial resistance patterns for both strains. Bacterial isolates with multiple antimicrobial resistance were recovered in both treatments, for both kinds of strains, and during the entire period of the experiment (data not shown). Table 1. Evaluation of the E. coli number and temperature during composting in aerated (Treatment A) and non-aerated (Treatment B) systems. Each data is the mean of the six equidistant points inside the heaps Week Composting A (aerated) CFU/mL Temperature (oC) Composting B (non-aerated) CFU/mL Temperature(oC) 1 934 58 1234 47 2 540 61 935 51 3 510 66 624 61 4 278 34 178 35 5 34 19 23 22 6 21 22 15 21 7 14 14 14 14 Figure1 A, B. Fate of E. coli strains during composting. A: aerated treatment, B: non- aerated treatment. Table 2. Virulence genes of E. coli isolates obtained from an ovine manure-based composting (n= 695 isolates) Number of isolates genotype Composting A (aerated) Composting B (non-aerated) stx 1 16 26 stx 1/ eae 1 16 stx 2 40 45 stx 2/ eae 47 stx 1/ stx 2 46 stx 1/ stx 2/ eae 1 7 non-STEC 255 267 Total 321 374 1774 Arq. Bras. Med. Vet. Zootec., v.66, n.6, p.1771-1778, 2014 Fate of non O157 . Amp-ampicillin; Tet-tetracycline; Tri-trimethoprim; Eri-erythromycin; Nal-nalidixic acid; Est-streptomycin; Novnovobiocin; Lin-lincomycin; Pen-penicillin; Neo-neomycin Figure 2. Antimicrobial resistance patterns of 114 E. coli isolates all carrying stx 2 gene and 234 E. coli non-STEC strains, both from composting systems. DISCUSSION There is a growing public concern about the link between livestock production and soil contamination by pathogenic bacteria. This is especially true for the application of raw manure, which is potentially capable of spreading pathogens to a wider environment (Turner, 2002; Nicholson et al., 2005; Bolton et al., 2011). Some foodborne disease outbreaks have been associated with manure contamination of fresh produce with STEC strains, especially O157:H7 (Islam et al., 2005) but also with non-O157 strains (Bolton et al., 2011). Sheep harbor many serotypes of STEC in their gastrointestinal tract; many of them associated with disease in humans and spread them to the environment by fecal shedding (Kudva et al., 1998; Vettorato et al., 2003). Although the elimination of pathogens by composting has been well documented (Larney et al., 2003; Nicholson et al., 2005), composting time and temperatures required to achieve elimination or reduction of the number of E. coli vary widely. Turner (2002) demonstrated inactivation of E. coli in farmyard manure after 2 hours at 55oC. Lung et al. (2001) described an effectively reduction in the E. coli O157:H7 number after 4 days at 45oC. In contrast, Nicholson et al. (2005) reported the recuperation of E. coli O157 from manure heaps after 32 days of composting. In the present study the suitable temperature for the reduction or elimination of the pathogenic microorganisms for both composting treatments was reached around the first and the second week respectively for aerated and non-aerated treatments, which agrees partially with the data reported by Nicholson et al. (2005) E. coli commensal isolates could be recovered from the composting systems until the seventh week, while the non-O157 STEC strains carrying stx1 or stx2 were isolated for 49 days in both composting treatments. There have been few studies on the persistence of E. coli non-O157:H7 in animal feces. Gonçalves and Marin (2007) described STEC non-O157 survival for 25 and 30 days at 40oC and 38oC respectively in a bovine manure-based composting. Fremaux et al. (2007) reported the elimination of non-O157 cells submitted to composting in manure heaps after nine and 16 days at 65oC and 56oC respectively, indicating that temperature is a key factor in the elimination and/or reduction of the viable cells. Not only temperature but also the interactions among the different types of bacteria and fungi inside of the composting system is important (Himathongkham and Rieman, 1999; Turner, 2002; Larney et al., 2003) which could enfocar la importancia de la producción mediática de los niños en su descubrimiento del mundo, sobre todo utilizando el periódico escolar y la imprenta. Asimismo las asociaciones de profesores trabajaron en esta línea e incluso la enseñanza católica se comprometió desde los años sesenta realizando trabajos originales en el marco de la corriente del Lenguaje Total. Páginas 43-48 45 Comunicar, 28, 2007 En el ámbito de los medios, también desde el principio del siglo XX hay ciertas corrientes de conexión. Pero es a lo largo de los años sesenta cuando se constituyeron asociaciones de periodistas apasionados por sus funciones de mediadores, que fomentaron la importancia ciudadana de los medios como algo cercano a los jóvenes, a los profesores y a las familias. Así se crearon la APIJ (Asociación de Prensa Información para la Juventud), la ARPEJ (Asociación Regional de Prensa y Enseñanza para la Juventud), el CIPE (Comité Interprofesional para la Prensa en la Escuela) o la APE (Asociación de Prensa y Enseñanza), todas ellas para la prensa escrita Estas asociaciones fueron precedidas por movimientos surgidos en mayo de 1968, como el CREPAC que, utilizando películas realizadas por periodistas conocidos, aclaraba temas que habían sido manipulados por una televisión demasiado próxima al poder político y realizaba encuentros con grupos de telespectadores. cipio del siglo XX, y nos han legado textos fundadores muy preciados, importantes trabajos de campo y muchos logros educativos y pedagógicos. La educación en medios ha tenido carácter de oficialidad de múltiples maneras, aunque nunca como una enseñanza global. Así la campaña «Operación Joven Telespectador Activo» (JTA), lanzada al final de los años setenta y financiada de manera interministerial para hacer reflexionar sobre las prácticas televisuales de los jóvenes, la creación del CLEMI (Centro de Educación y Medios de Comunicación) en el seno del Ministerio de Educación Nacional en 1983, la creación de la optativa «Cine-audiovisual» en los bachilleratos de humanidades de los institutos en 1984 (primer bachillerato en 1989) y múltiples referencias a la educación de la imagen, de la prensa, de Internet. La forma más visible y rápida de evaluar el lugar de la educación en medios es valorar el lugar que se le ha reservado en los libros de texto del sistema educa- 2. Construir la educación en los medios sin nombrarla El lugar que ocupa la edu- La denominación «educación en medios», que debería cación en los medios es muy ambiguo, aunque las cosas están cambiando recientemente. entenderse como un concepto integrador que reagrupase todos los medios presentes y futuros, es a menudo percibida En principio, en Francia, co- por los «tradicionalistas de la cultura» como una tendencia mo en muchos otros países, la educación en los medios no es hacia la masificación y la pérdida de la calidad. una disciplina escolar a tiempo completo, sino que se ha ido conformado progresivamente a través de experiencias y reflexiones teóricas que han tivo en Francia. Una inmersión sistemática nos permi- permitido implantar interesantes actividades de carác- te constatar que los textos oficiales acogen numerosos ter puntual. Se ha ganado poco a poco el reconoci- ejemplos, citas, sin delimitarla con precisión. miento de la institución educativa y la comunidad es- colar. Podemos decir que ha conquistado un «lugar», 3. ¿Por qué la escuela ha necesitado casi un siglo en el ámbito de la enseñanza transversal entre las dis- para oficilializar lo que cotidianamente se hacía en ciplinas existentes. ella? Sin embargo, la escuela no está sola en esta aspi- Primero, porque las prácticas de educación en me- ración, porque el trabajo en medios es valorado igual- dios han existido antes de ser nombradas así. Recor- mente por el Ministerio de Cultura (campañas de foto- demos que no fue hasta 1973 cuando aparece este grafía, la llamada «Operación Escuelas», presencia de término y que su definición se debe a los expertos del colegios e institutos en el cine ), así como el Minis- Consejo Internacional del Cine y de la Televisión, que terio de la Juventud y Deportes que ha emprendido en el seno de la UNESCO, definen de esta forma: numerosas iniciativas. «Por educación en medios conviene entender el estu- Así, esta presencia de la educación en los medios dio, la enseñanza, el aprendizaje de los medios moder- no ha sido oficial. ¡La educación de los medios no apa- nos de comunicación y de expresión que forman parte rece oficialmente como tal en los textos de la escuela de un dominio específico y autónomo de conocimien- francesa hasta 2006! tos en la teoría y la práctica pedagógicas, a diferencia Este hecho no nos puede dejar de sorprender ya de su utilización como auxiliar para la enseñanza y el que las experiencias se han multiplicado desde el prin- aprendizaje en otros dominios de conocimientos tales Páginas 43-48 46 Comunicar, 28, 2007 como los de matemáticas, ciencias y geografía». A pe- mente en todas las asignaturas. Incluso los nuevos cu- sar de que esta definición ha servido para otorgarle un rrículos de materias científicas en 2006 para los alum- reconocimiento real, los debates sobre lo que abarca y nos de 11 a 18 años hacen referencia a la necesidad no, no están totalmente extinguidos. de trabajar sobre la información científica y técnica y En segundo lugar, porque si bien a la escuela fran- el uso de las imágenes que nacen de ella. cesa le gusta la innovación, después duda mucho en Desde junio de 2006, aparece oficialmente el tér- reflejar y sancionar estas prácticas innovadoras en sus mino «educación en medios» al publicar el Ministerio textos oficiales. Nos encontramos con una tradición de Educación los nuevos contenidos mínimos y las sólidamente fundada sobre una transmisión de conoci- competencias que deben adquirir los jóvenes al salir mientos muy estructurados, organizados en disciplinas del sistema educativo. escolares que se dedican la mayor parte a transmitir Este documento pretende averiguar cuáles son los conocimientos teóricos. La pedagogía es a menudo se- conocimientos y las competencias indispensables que cundaria, aunque los profesores disfrutan de una ver- deben dominar para terminar con éxito su escolaridad, dadera libertad pedagógica en sus clases. El trabajo seguir su formación y construir su futuro personal y crítico sobre los medios que estaba aún en elaboración profesional. Siete competencias diferentes han sido te- necesitaba este empuje para hacerse oficial. nidas en cuenta y en cada una de ellas, el trabajo con Aunque el trabajo de educación en los medios no los medios es reconocido frecuentemente. Para citar esté reconocido como disciplina, no está ausente de un ejemplo, la competencia sobre el dominio de la len- gua francesa definen las capa- cidades para expresarse oral- La metodología elaborada en el marco de la educación en mente que pueden adquirirse con la utilización de la radio e, medios parece incluso permitir la inclinación de la sociedad incluso, se propone fomentar de la información hacia una sociedad del conocimiento, como defiende la UNESCO. En Francia, se necesitaría unir el interés por la lectura a través de la lectura de la prensa. La educación en los medios las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio adquiere pleno derecho y entidad en la sección sexta titulada «competencias sociales y cívi- técnico de los aparatos. cas» que indica que «los alum- nos deberán ser capaces de juz- gar y tendrán espíritu crítico, lo que supone ser educados en los las programaciones oficiales, ya que, a lo largo de un medios y tener conciencia de su lugar y de su influencia estudio de los textos, los documentalistas del CLEMI en la sociedad». han podido señalar más de una centena de referencias a la educación de los medios en el seno de disciplinas 4. Un entorno positivo como el francés, la historia, la geografía, las lenguas, Si nos atenemos a las cifras, el panorama de la las artes plásticas : trabajos sobre las portadas de educación en medios es muy positivo. Una gran ope- prensa, reflexiones sobre temas mediáticos, análisis de ración de visibilidad como la «Semana de la prensa y publicidad, análisis de imágenes desde todos los ángu- de los medios en la escuela», coordinada por el CLE- los, reflexión sobre las noticias en los países europeos, MI, confirma año tras año, después de 17 convocato- información y opinión rias, el atractivo que ejerce sobre los profesores y los Esta presencia se constata desde la escuela mater- alumnos. Concebida como una gran operación de nal (2 a 6 años) donde, por ejemplo, se le pregunta a complementariedad source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. Ruth Rousing, Hanne Willumsen, Carsten Nielsen and Flemming Jessen are thanked for excellent technical help. The Danish HIV-Cohort is thanked for providing us HIV-related data. 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(2010) Serum FGF21 levels are elevated in association with lipodystrophy, insulin resistance and biomarkers of liver injury in HIV-1-infected patients. AIDS 24: 2629–2637. PLOS ONE | www.plosone.org 7 March 2013 | Volume 8 | Issue 3 | e55632
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