Dentre as espécies conhecidas do gênero Arcobacter, as espécies A. butzleri, A. cryaerophilus e A. skirrowii são consideradas potecialmente zoonóticas, podendo ser transmitidas por alimentos de origem animal. O presente estudo teve como objetivos isolar e caracterizar fenotipica e genotipicamente cepas de Arcobacter spp. provenientes de carcaças de suínos e amostras de ambiente de abatedouro localizado no Estado de São Paulo. As cepas isoladas foram submetidas a reação em cadeia pela polimerase para a identificação e detecção de um grupo de genes de virulência. A concentração inibitória mínima frente a nove antimicrobianos usados para o controle da infecção pelo agente foi determinada e as cepas foram analisadas através do PFGE e pelo AFLP. Dentre as 30 carcaças avaliadas, 25 foram positivas para o agente e 70 cepas foram selecionadas e identificadas como Arcobacter spp. As espécies isoladas foram A. butzleri (n=61), A. cryaerophilus (n=7) e A. skirrowii (n=2). A frequência dos possíveis genes de virulência encontrada variou de 71,4% a 100% para os genes tlyA, pldA, cj1349, ciaB, cadF e mviN. Não foram detectados os genes hecA, hecB e irgA. O perfil de virulência ciaB/ cj1349/ mviN/ cadF/ pldA/ tlyA foi o mais frequente e detectado em 66% das cepas. Todas as cepas foram sensíveis à gentamicina e tetraciclina e 77,1% foram multirresistentes, dentre estas o perfil mais frequente foi de resistência a azitromicina/ florfenicol/ ácido nalidíxico/ telitromicina/ clindamicina Houve grande diversidade genotípica entre as cepas através do PFGE e do AFLP, e ambas a técnicas apresentaram o mesmo poder discriminatório na análise das cepas isoladas. / Among the known species of the genus Arcobacter, the species A. butzleri, A. cryaerophilus and A. skirrowii are considered potentially zoonotic and can be transmitted by food of animal origin. This study aimed to isolate and characterize phenotypically and genotypically strains of Arcobacter spp from swine carcasses and slaughterhouse environment samples located in the State of São Paulo. The isolated strains were subjected to polymerase chain reaction for identification and detection of a group of putative virulence genes. The minimum inhibitory concentration was determined against nine antimicrobials indicated for the control of infection by the agent and the strains were analyzed by PFGE and by AFLP. Among the 30 carcasses evaluated, 25 were positive for the agent and 70 strains were selected and identified as Arcobacter spp. The isolated species were A. butzleri (n = 61), A. cryaerophilus (n = 7) and A. skirrowii (n = 2). The frequency of virulence genes found ranged from 71.4 % to 100 % for genes tlyA , pldA , cj1349 , ciaB , cadF and mviN . The genes hecA, hecB and irgA were not detected. The virulence profile ciaB/ cj1349/ mviN/ cadF/ pldA/ tlyA was the most frequent and detected in 66 % of the strains. All strains were susceptible to gentamicin and tetracycline and 77.1% were multirresistant, among these the most common profile of resistance was azithromycin/ florfenicol/ nalidixic acid/ telithromycin/ clindamycin There were large genotypic diversity among strains by PFGE and AFLP and both techniques showed the same discriminatory power in the analysis of the isolated strains.

Arcobacter spp. é um micro-organismo Gram negativo que provoca diarreia aquosa e sepse em seres humanos. A. butzleri, A. cryaerophilus e A. skirrowii são espécies patogênicas para humanos. O objetivo deste estudo foi detectar a presença de Arcobacter spp. na carne de aves comercializadas em açougues na cidade de São Paulo, verificando os genes de virulência e o perfil genotípico. Um total de 300 cortes de carne de frango foram submetidos ao cultivo e isolamento sob condições aeróbicas, a 30°C por 72 horas. Colônias suspeitas de Arcobacter spp. foram selecionadas para a detecção molecular pela reacção em cadeia da polimerase (PCR), a fim de determinar as espécies e os genes de virulência. Os resultados revelaram a presença de Arcobacter spp. em 18.3% (55/300) de amostras de carne de aves, sendo identificado como A. butzleri 63,6% (35/55) e A. cryaerophilus 36,3% (20/55). Os genes de virulência pesquisados demonstraram positividade de 100% (55/55) para o ciaB e mviN, seguidos de cj1349 98,1% (54/55), pldA 94,4% (52/55), cadF 72,7% (40/55), tlyA 92,7% (51/55), hecA 49% (27/55), irgA 47,2% (26/55) e hecB 34,5% (19/55). Estas cepas foram submetidas ao AFLP gerando dois dendogramas. Foram identificados 19 perfis genotípicos para A. butzleri e 17 para A. cryaerophilus. Os resultados desta pesquisa apontam a presença de A. butzleri e A. cryaerophilus na fase final da distribuição de carne de frangos nos açougues. A falta de inocuidade dos alimentos de origem animal, bem como a presença de estirpes virulentas representam riscos de Saúde Pública, com especial atenção para a possibilidade de contaminação cruzada gerados por alimentos crus e utensílios de cozinha / Arcobacter spp. is a Gram negative microorganism that causes watery diarrhea and septicemia in humans. A. butzleri, A. cryaerophilus and A. skirrowii are pathogenic for humans. The aim of this study was to detect the presence of Arcobacter spp. in poultry meat from butcher shops in the city of São Paulo, checking the virulence genes and profile genotypic. A total of 300 chicken cuts were used for cultivation and isolation in broth and JM agar, under aerobic conditions, at 30 °C for 72 hours. Arcobacter colonies were selected and submitted to molecular detection by polymerase chain reaction (PCR), in order to determine species and virulence genes. Results show the presence of Arcobacter spp. in 18.3% (55/300) of poultry meat samples, identified as A. butzleri 63.6% (35/55) and A. cryaerophilus 36.3% (20/55) in chicken isolates. The virulence genes on Arcobacter spp. researched demonstrated positive these 100% (55/55) for ciaB and mviN, followed by cj134998,1% (54/55), pldA 94,4% (52/55), cadF 72.7% (40/55), tlyA 92,7% (51/55), hecA 49% (27/55), irgA 47,2% (26/55) and hecB 34,5% (19/55). These strains were submitted to AFLP generating two dendogramas. Nineteen profiles genotypins were obtained for A. butzleri and seventeen for A. cryaerophilus. The results of this research point to the presence of Arcobacter butzleri and A. cryaerophilus in the final stage of distribution of poultry meat to butcher shops. The lack of safety of food of animal origin, as well as the presence of virulent strains pose risks to Public Health, with special focus on cross contamination risks generated by uncooked food and utensils

Microbial fuel cells (MFCs) are electrochemical devices that exploit the ability of certain microorganisms to anaerobically respire using an insoluble terminal electron acceptor and therefore generate an electrical current. These bacteria are called electrogens or electrogenic bacteria. Two species of Arcobacter, Arcobacter butzleri ED-1 and Arcobacter L were isolated from the anodic chamber of an acetate fed MFC, and A. butzleri ED-1 was found to be the more electrogenic of the two bacteria. Arcobacter spp. are  proteobacteria and A. butzleri ED-1 and Arcobacter L were the first example of electrogenically active e proteobacteria. It was decided to study their interactions with the anode by fluorescent microscopy and study their electrogenic mechanisms by comparative proteomics using the iTRAQ method as it would allow for simultaneous identification and quantification of peptides in multiple samples. Fluorescent imaging over a period of 120 h in a half MFC showed that both A. butzleri ED-1 and Arcobacter L formed a thin anodic biofilm of a few cells thick and that A. butzleri ED-1 maintained a more stable anodic biofilm than Arcobacter L. iTRAQ analysis showed that the flagellin FlaA was up-regulated 2.4 fold at the anode but no other electron transport proteins or adhesins were upregulated. These results were distinct from those observed for other electrogenic bacteria (Geobacter sulfurreducens and Shewanella oneidensis MR-1) in previous studies which exhibited up-regulated electron transport proteins at the anode as well as forming an anodic biofilm of 50 μm thick. Therefore based on these results it was concluded that FlaA was most likely playing an important role A. butzleri ED-1 anode biofilm formation and that the mechanisms of electrogenesis in A. butzleri ED- 1 and Arcobacter L may be novel compared to those previously characterised. It was also concluded that one possible reason for A. butzleri ED-1 being more electrogenic than Arcobacter L was its ability to form a more stable anodic biofilm. It must be noted that both of these conclusions are highly speculative and further study is needed to elucidate the electrogenic mechanisms of A. butzleri ED-1 and to further compare biofilm formation between the two species.

Twenty species and six subspecies of the genera Arcobacter and Campylobacter have been described to date. All are Gram-negative, microaerophilic, curved, spiral or S-shaped cells, and are members of the order Campylobacterales, class Epsilonproteobacteria phylum Proteobacteria. Though most members are pathogenic, C. jejuni, C. coli and A. butzleri are the most frequently isolated species from patients suffering from gastrointestinal illness. The current methods for their detection, identification, and differentiation are cumbersome, time consuming and lack specificity. DNA based molecular techniques including real-time Polymerase Chain Reaction (PCR) and Fingerprinting methods Terminal Restriction Fragments Length Polymorphism (T-RFLP) and Ligase Detection Reaction (LDR) have been used in this project to develop rapid detection and identification methods for Campylobacter and Arcobacter species. Five real-time PCR methods were developed which include: (a) rapid detection and identification of Campylobacter species using real-time PCR adjacent hybridisation probes, (b) rapid identification of C. jejuni using SYBR Green I, (c) rapid detection and differentiation of Arcobacter species using adjacent hybridisation probes, (d) rapid detection and differentiation of Arcobacter species and the Campylobacter group (C. coli, C. jejuni, C. lari, C. hyoilei, C. helviticus, C. hyointestinalis, C. insulaenigrae, C lanienae) using melting temperature (Tm) of adjacent hybridisation probes, and (e) a one tube real-time PCR multiplex for the rapid detection and identification of Campylobacter species, C. coli and C. jejuni using a TaqMan Probe, in an iCycler iQTM (BioRad, USA) and Light CyclerTM (Idaho Technology, USA). [Continued ...]

Pyruvate:ferredoxin oxidoreductase activity was demonstrated in microaerophilic members of Campylobacter, Wolinella, and Helicobacter. Arcobacter cryaerophila (sic) and Arcobacter nitrofigilis, two aerobic species, lacked any detectable activity. Under anaerobic conditions crude extracts of Campylobacter, Helicobacter and Wolinella were capable of reducing the electron carriers benzyl viologen and metronidazole in the presence of pyruvate. Addition of Clostridium pasteurianum ferredoxin to the metronidazole-linked reaction enhanced metronidazole-reducing activity, suggesting that electron transport to artificial electron carriers is facilitated by ferredoxin. All species exhibited varying degrees of sensitivity to metronidazole (MIC = <0.8 to 25 µg/ml) except Arcobacter cryaerophila, which was resistant to > 100 µg/ml. This further supports the theory that these organisms possess ferredoxin-linked reactions. The presence of the oxygen-labile enzyme pyruvate:ferredoxin oxidoreductase may be related to the inability of these microaerophilic bacteria to grow in normal atmospheric levels of oxygen. Under aerobic conditions crude extracts of the organisms were also capable of reducing NAD in the presence of pyruvate. This might be accounted for by an NAD-linked pyruvate dehydrogenase; alternatively, it might be due to an enzymatic reduction of NAD by electrons from the reduced ferredoxin generated during the ferredoxin-linked pyruvate oxidoreductase reaction. / Master of Science

<p>Species of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals / and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42&ordm / C as well as at ambient room temperature (&plusmn / 22&ordm / C). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4oC for up to 297 days.</p>

<p>Species of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals / and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42&ordm / C as well as at ambient room temperature (&plusmn / 22&ordm / C). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4oC for up to 297 days.</p>

Zugl.: Berlin, Freie Universiẗat, Diss., 2008. / Includes bibliographical references.

Philosophiae Doctor - PhD / Species of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals; and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42ºC as well as at ambient room temperature (±222ºC). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4ºC for up to 297 days. / South Africa

Bacteria of the Arcobacter genus are microorganisms belonging to the Campylobacteraceae family. Currently, the Arcobacter genus comprises 23 species, isolated from a variety of hosts and ecological niches. Although most of the pathogenic species are from fecal origin, seawater is considered to be the habitat for most of them. The transmission to humans seems to be associated to the consumption of raw food or not entirely-cooked. In this regard, the presence of pathogenic Arcobacter species in food of marine origin and vegetables has not been deeply studied. When the detection and identification of Arcobacter is performed exclusively using conventional culture-based methods, the process is slow, tedious and rarely effective many times, originating frequently false negatives. Molecular biology-based methods analyzing nucleic acids with the use of the Polymerase Chain Reaction (PCR), are alternative procedure characterized by being reliable, faster, more sensitive. In this thesis we have analysed the presence of Arcobacter spp in 100 samples from shellfish and another 100 samples from vegetables using both approaches, on one side through the isolation and characterization by plate culture in and on the other side by PCR. The results obtained by both methods confirm the presence of Arcobacter spp. in the samples. An enrichment period followed by PCR has proved to be more sensitive than culture for the detection of the microorganism in the samples. In this study, Arcobacter contamination has been detected in 71 % of the shellfish samples and 20 % of vegetable samples. The obtained Arcobacter isolates have been identified to the species level analyzing the 16 rRNA gene by PCR-RFLP. This technique has allowed the identification of the species A. butzleri, A. cryaerophilus and A. defluvii in the shellfish isolates, being the first time that A. defluvii is identified in clams. Additionally, it is also the first time that Arcobacter spp. is detected in cockles. A. butzleri and A. cryaerophilus have been isolated in vegetables being the last one the first time in this type of samples. The sensitivity of the obtained isolates to ciprofloxacin and levofloxacin has also been studied. 2 % in analysed shellfish and 1 % in vegetables were contaminated with resistant strains. Three isolates from shellfish and 2 from vegetables, previously identified as A. butzleri, have shown resistance to both fluoroquinolones. In all of them, a mutation in the 254 position (C normal to T mutant) in the Quinolone Resistance-Determining Region (QRDR) of the gyrA gene has been detected. Our results regarding the presence of the pathogen in both types of samples are sufficiently relevant as to consider that the consumption of these contaminated foods with Arcobacter, especially A. butzleri, could suppose a risk for human health. In addition, in this thesis the analisys of Helicobacter spp. and H. pylori by conventional PCR and real-time PCR has been carried out. The Helicobacter genus comprises a large number of species considered pathogenic. The most studied specie is H. pylori, one of the most common pathogens in humans and responsible of 90 % of peptic ulcers and closely related to the development of gastric cancer. Although it seems clear that H. pylori is transmited by fecal-oral route through the water, its presence in food is poorly known. Therefore, another objective of the present study has been to study the presence of these organisms in shellfish and vegetable samples. Helicobacter spp. or H. pylori could not be detected by conventional PCR. However, the real-time PCR technique allowed the detection of H. pylori in a sample from vegetables (lettuce). / Las bacterias del género Arcobacter son microorganismos pertenecientes a la familia Campylobacteraceae. Actualmente, el género Arcobacter comprende 23 especies, aisladas de una gran diversidad de hospedadores y nichos ecológicos. Las aguas de mar son consideradas el hábitat natural para muchas de ellas, aunque la mayor parte de las especies patógenas son de origen fecal. La transmisión al hombre parece estar asociada al consumo de alimentos crudos o poco cocidos. Sin embargo, la presencia de especies patógenas de Arcobacter en alimentos de origen marino y vegetales ha sido muy poco estudiada. Cuando la detección e identificación de Arcobacter, se establece exclusivamente en función de métodos convencionales de cultivo, el proceso resulta lento, tedioso y poco efectivo en muchas ocasiones, originando frecuentes falsos negativos. Los métodos de detección molecular basados en el análisis de ácidos nucleicos, como la Reacción en Cadena de la Polimerasa (PCR), pueden suponer una alternativa más rápida, sensible y fiable. Por todo ello, en este trabajo se ha realizado la detección de Arcobacter spp. mediante aislamiento por cultivo en placa y PCR, a partir de 100 muestras de moluscos y 100 de verduras. Los resultados obtenidos por ambos métodos confirman la existencia de Arcobacter en las muestras. La PCR, realizada tras un periodo de enriquecimiento, ha resultado ser más sensible que el cultivo para la detección del microorganismo en las muestras. En este trabajo se ha detectado contaminación por Arcobacter en el 71 % de las muestras de moluscos y el 20 % de las muestras de verduras. Los aislados de Arcobacter obtenidos han sido identificados a nivel de especie mediante un análisis PCR-RFLP del gen 16 ARNr. Esta técnica ha permitido identificar las especies A. butzleri, A. cryaerophilus y A. defluvii de los aislados de moluscos, siendo la primera vez que es identificada A. defluvii de muestras de almejas. También es la primera vez que se detecta Arcobacter spp. en berberechos. En el caso de las verduras, se han aislado A. butzleri y A. cryaerophilus. Esta última especie ha sido identificada por primera vez en este tipo de muestras. También se ha estudiado la sensibilidad de los aislados obtenidos a ciprofloxacino y levofloxacino. El porcentaje de muestras contaminadas con cepas resistentes fue del 2 % en moluscos y del 1 % en verduras. Tres aislados procedentes de moluscos y 2 de verduras, identificados previamente como A. butzleri, han mostrado resistencia a ambas fluoroquinolonas. En todos ellos se ha detectado la existencia de una mutación en la posición 254 (C normal por T mutante) en la Región Determinante de Resistencia a Quinolonas (QRDR) del gen gyrA. Nuestros resultados sobre la presencia del patógeno en ambos tipos de muestras son lo suficientemente relevantes como para considerar que el consumo de estos alimentos contaminados con Arcobacter, especialmente A. butzleri, podría suponer un riesgo para la salud humana. Adicionalmente, en este trabajo se ha realizado un análisis de detección de Helicobacter spp. y H. pylori mediante PCR convencional y PCR a tiempo real. El género Helicobacter comprende un gran número de especies consideradas patógenas. La especie más estudiada es H. pylori, uno de los patógenos más comunes en humanos, responsable del 90 % de las úlceras pépticas y relacionado estrechamente con el desarrollo de cáncer gástrico. Aunque parece claro que H. pylori se transmite por la vía fecal-oral a través del agua, su presencia en los alimentos es poco conocida. Por lo tanto, en este estudio se tomó como objetivo estudiar la presencia de estos microorganismos en las muestras de moluscos y verduras. El análisis mediante PCR convencional no mostró resultados positivos para la detección de Helicobacter spp. ni H. pylori. La técnica PCR a tiempo real, sin embargo, ha permitido la detección de H. pylori en una muestra procedente de verduras (lechuga). / Els bacteris del gènere Arcobacter són microorganismes pertanyents a la família Campylobacteraceae. Actualment, el gènere Arcobacter comprèn 23 espècies, aïllades d'una gran diversitat d'hostes i nínxols ecològics. Les aigües de la mar són considerades l'hàbitat natural per a moltes d'aquestes espècies, encara que la major part de les patògenes són d'origen fecal. La transmissió a l'home sembla estar associada al consum d'aliments crus o poc cuits. No obstant això, la presència d'espècies patògenes d'Arcobacter en aliments d'origen marí i vegetals ha sigut molt poc estudiada. Quan la detecció i identificació d'Arcobacter s'estableix exclusivament en funció de mètodes convencionals de cultiu, el procés resulta lent, tediós i sovint poc efectiu, i origina amb freqüència falsos negatius. Els mètodes de detecció molecular basats en l'anàlisi d'àcids nucleics, com la reacció en cadena de la polimerasa (PCR), poden suposar una alternativa més ràpida, sensible i fiable. Per tot això, en aquest treball s'ha realitzat la detecció d'Arcobacter spp. mitjançant aïllament per cultiu en placa i PCR, a partir de 100 mostres de mol·luscos i 100 de verdures. Els resultats obtinguts pels dos mètodes confirmen l'existència d'Arcobacter en les mostres. La PCR, després d'un període d'enriquiment, ha resultat ser més sensible que el cultiu per a la detecció del microorganisme en mostres de mol·luscos. En aquest treball, s'ha detectat contaminació per Arcobacter en el 71 % de les mostres de mol·luscos i el 20 % de les mostres de verdures. Els aïllats d'Arcobacter obtinguts han sigut identificats a nivell d'espècie mitjançant una anàlisi PCR-RFLP del gen 16 ARNr. Aquesta tècnica ha permès identificar les espècies A. butzleri, A. cryaerophilus i A. defluvii en els aïllats de mol·luscos. Aquesta és la primera vegada que A. defluvii és identificada en mostres de cloïsses. També és la primera vegada que es detecta Arcobacter spp. en catxels. En el cas de les verdures, s'han aïllat A. butzleri i A. cryaerophilus. Aquesta última espècie ha sigut identificada per primera vegada en aquest tipus de mostres. També s'ha estudiat la sensibilitat dels aïllats obtinguts a la ciprofloxacina i la levofloxacina. El percentatge de mostres contaminades amb soques resistents va ser del 2 % en mol·luscos i de l'1 % en verdures. 3 aïllats procedents de mol·luscos i 2 de verdures, identificats prèviament com A. butzleri, han mostrat resistència a ambdues fluoroquinolones. En tots aquests s'ha detectat l'existència d'una mutació en la posició 254 (C normal per T mutant) en la regió determinant de resistència a quinolones (QRDR) del gen gyrA. Els nostres resultats sobre la presència del patogen en els dos tipus de mostres són prou rellevants per a considerar que el consum d'aquests aliments contaminats amb Arcobacter, especialment A. butzleri, podria suposar un risc per a la salut humana. Addicionalment, en aquest treball s'ha fet una anàlisi de detecció d'Helicobacter spp. i H. pylori mitjançant PCR convencional i PCR a temps real. El gènere Helicobacter comprèn un gran nombre d'espècies considerades patògenes. L'espècie més estudiada és H. pylori, un dels patògens més comuns en humans, responsable del 90% de les úlceres pèptiques i relacionada estretament amb el desenvolupament de càncer gàstric. Encara que sembla clar que H. pylori es transmet per la via fecal-oral a través de l'aigua, la presència d'aquest bacteri en els aliments és poc coneguda. Per tant, en aquest estudi es va definir com a objectiu estudiar la presència d'aquests microorganismes en les mostres de mol·luscos i verdures. L'anàlisi mitjançant PCR convencional no va mostrar resultats positius per a la detecció d'Helicobacter spp. ni d'H. pylori. La tècnica PCR a temps real, en canvi, ha permès la detecció d'H. pylori en una mostra procedent de verdures (encisam). / Bayas Morejón, IF. (2016). Aportaciones a la epidemiología de Arcobacter y Helicobacter spp.: Aplicación de métodos moleculares a su detección e identificación en alimentos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/75087 / TESIS