Arcobacter butzleri,Arcobacter cryaerophilus,and Arcobacter skirrowii Circulation in a Dairy Farm and Sources of Milk Contamination

Even though dairy cows are known carriers of Arcobacter species and raw or minimally processed foods are recognized as the main sources of human Arcobacter infections in industrialized countries, data on Arcobacter excretion patterns in cows and in milk are scant. This study aimed to identify potentially pathogenic Arcobacter species in a dairy herd and to investigate the routes of Arcobacter transmission among animals and the potential sources of cattle infection and milk contamination. A strategy of sampling the same 50 dairy animals, feed, water, and milk every month for a 10-month period, as well as the sampling of quarter milk, animal teats, the milking environment, and animals living on the farm (pigeons and cats), was used to evaluate, by pulsed-field gel electrophoresis (PFGE), the characteristic patterns in animals, their living environment, and the raw milk they produced. Of the 463 samples collected, 105 (22.6%) were positive for Arcobacter spp. by culture examination. All the matrices except quarter milk and pigeon gut samples were positive, with prevalences ranging from 15 to 83% depending on the sample. Only three Arcobacter species, Arcobacter cryaerophilus (54.2%), A. butzleri (34.2%), and A. skirrowii (32.3%), were detected. PFGE analysis of 370 isolates from positive samples provided strong evidence of Arcobacter circulation in the herd: cattle likely acquire the microorganisms by orofecal transmission, either by direct contact or from the environment, or both. Water appears to be a major source of animal infection. Raw milk produced by the farm and collected from a bulk tank was frequently contaminated (80%) by A. butzleri; our PFGE findings excluded primary contamination of milk, whereas teats and milking machine surfaces could be sources of Arcobacter milk contamination.     

Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.     

Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens.

The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.     

Induction and resuscitation of viable nonculturable Arcobacter butzleri cells

Two strains of Arcobacter butzleri, ATCC 49616 and an environmental isolate, became nonculturable in seawater microcosms at 4 degrees C by 20 days and at room temperature by 14 days. Nonculturable cells were viable for up to 270 days of incubation in microcosms. Resuscitation of A. butzleri cells from microcosms at both temperatures was achieved 9 days after nutrient addition.     

黄柏果实提取物对植物病原真菌的抑制作用

在室内测定了芸香科植物黄柏(Phellodendron chinese Schneid.)果实乙醇粗提物及其4个不同极性溶剂的萃取部分在浓度为1 mg/mL时对小麦纹枯病菌(Rhizoctonia cerealis)、稻纹枯病菌(Rhizoctonia solani)、番茄镰刀菌萎焉病菌(Fusarium oxysporum f. sp.lycopersici)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum)、棉花枯萎病菌(Fusarium vasinfectum)、棉花黄萎病菌(Verticillium dahliae)、小麦赤霉病菌(Fusarium graminearum)、玉米小斑病菌(Bipolaris maydis)、西瓜枯萎病菌(Fusarium oxysporum f.sp.niverum)、梨黑星病菌(Venturia piri-na)、稻瘟病菌(Magnaporthe grisea)等11种植物病原真菌的抑制作用.乙酸乙酯部分和正丁醇部分对植物病原菌均表现出较强的抑制活性,其中乙酸乙酯部分对两种丝核菌小麦纹枯病菌和稻纹枯病菌的生长抑制作用最强,抑制率分别为100.00%和89.36%;正丁醇部分对两者的抑制率分别为97.32%和61.32%.实验结果表明,黄柏果实中的抗真菌活性成分主要存在于乙醇粗提物中的乙酸乙酯和正丁醇萃取部分中.     

Complete genome sequences of Arcobacter butzleri ED-1 and Arcobacter sp. strain L, both isolated from a microbial fuel cell

Arcobacter butzleri strain ED-1 is an exoelectrogenic epsilonproteobacterium isolated from the anode biofilm of a microbial fuel cell. Arcobacter sp. strain L dominates the liquid phase of the same fuel cell. Here we report the finished and annotated genome sequences of these organisms.     

The complete genome sequence and analysis of the epsilonproteobacterium Arcobacter butzleri

Background

Arcobacter butzleri is a member of the epsilon subdivision of the Proteobacteria and a close taxonomic relative of established pathogens, such as Campylobacter jejuni and Helicobacter pylori. Here we present the complete genome sequence of the human clinical isolate, A. butzleri strain RM4018.

Methodology/Principal Findings

Arcobacter butzleri is a member of the Campylobacteraceae, but the majority of its proteome is most similar to those of Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, and those of the deep-sea vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. In addition, many of the genes and pathways described here, e.g. those involved in signal transduction and sulfur metabolism, have been identified previously within the epsilon subdivision only in S. denitrificans, W. succinogenes, Sulfurovum, and/or Nitratiruptor, or are unique to the subdivision. In addition, the analyses indicated also that a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with a large number of respiration-associated proteins, signal transduction and chemotaxis proteins and proteins involved in DNA repair and adaptation. To investigate the genomic diversity of A. butzleri strains, we constructed an A. butzleri DNA microarray comprising 2238 genes from strain RM4018. Comparative genomic indexing analysis of 12 additional A. butzleri strains identified both the core genes of A. butzleri and intraspecies hypervariable regions, where <70% of the genes were present in at least two strains.

Conclusion/Significance

The presence of pathways and loci associated often with non-host-associated organisms, as well as genes associated with virulence, suggests that A. butzleri is a free-living, water-borne organism that might be classified rightfully as an emerging pathogen. The genome sequence and analyses presented in this study are an important first step in understanding the physiology and genetics of this organism, which constitutes a bridge between the environment and mammalian hosts.     

Genotyping and genetic diversity of Arcobacter butzleri by amplified fragment length polymorphism (AFLP) analysis

AIMS: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. METHODS AND RESULTS: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and North American origin from human infections, chickens, turkeys, ducks, sheep and poultry abbatoir effluent were studied by use of a protocol that involved stringent PCR amplification of fragments derived from digestion of genomic DNA with restriction enzymes BglII and Csp6I. The mean similarity value of duplicate profiles of 10 isolates was 91.15%, indicating the method to be reproducible. Numerical analysis of all 73 isolates distinguished 51 subtypes at the 91% similarity level, of which 39 comprised single strains. The remaining 34 isolates were distributed among 12 subtypes, each of which contained strains homogeneous with respect to their respective source of isolation. However, contemporaneous strains from the same source could also be distinguished. CONCLUSIONS: AFLP profiling is an effective method for typing the genetically diverse organism A. butzleri. SIGNIFICANCE AND IMPACT OF THE STUDY: The study represents a comprehensive analysis of the genetic diversity of A. butzleri by use of isolates from six countries spanning three continents and also shows that several distinct A. butzleri genotypes may be found in a given environment. AFLP profiling appears to have considerable potential for molecular epidemiological studies of this ubiquitous emerging pathogen that is implicated as a causative agent of both human and animal disease.     

Arcobacter butzleri in Sheep Ricotta Cheese at Retail and Related Sources of Contamination in an Industrial Dairy Plant

This study aimed to evaluate Arcobacter species contamination of industrial sheep ricotta cheese purchased at retail and to establish if the dairy plant environment may represent a source of contamination. A total of 32 sheep ricotta cheeses (1.5 kg/pack) packed in a modified atmosphere were purchased at retail, and 30 samples were collected in two sampling sessions performed in the cheese factory from surfaces in contact with food and from surfaces not in contact with food. Seven out of 32 samples (21.9%) of ricotta cheese collected at retail tested positive for Arcobacter butzleri at cultural examination; all positive samples were collected during the same sampling and belonged to the same batch. Ten surface samples (33.3%) collected in the dairy plant were positive for A. butzleri. Cluster analysis identified 32 pulsed-field gel electrophoresis (PFGE) patterns. The same PFGE pattern was isolated from more than one ricotta cheese sample, indicating a common source of contamination, while more PFGE patterns could be isolated in single samples, indicating different sources of contamination. The results of the environmental sampling showed that A. butzleri may be commonly isolated from the dairy processing plant investigated and may survive over time, as confirmed by the isolation of the same PFGE pattern in different industrial plant surface samples. Floor contamination may represent a source of A. butzleri spread to different areas of the dairy plant, as demonstrated by isolation of the same PFGE pattern in different production areas. Isolation of the same PFGE pattern from surface samples in the dairy plant and from ricotta cheese purchased at retail showed that plant surfaces may represent a source of A. butzleri postprocessing contamination in cheeses produced in industrial dairy plants.     

Fungicidal Activity of Some Common Weed Extracts Against Different Plant Pathogenic Fungi

Aqueous extract effects of 64 weed species on growth and development of Alternaria solani Sorauer. Helminthosporium satirum King & Bakke and Rhizodonia solani Kuhn. plant pathogenic fungi were studied in vitro . Extracts varied in the strength and persistence of their antifungal effects against the three fungi species. Some stimulated, others inhibited or had no effect. Among all species tested, extracts of Chenopodium murale, Falearia vulgaris. Ranunculus asiaticus and Sisymbrium irio were the most toxic to A. solani. Anagallis arlensis. Atriptex leucoclada, Crepis aspera. Notobasis syriaca. R. asiatieus, Rumex crispu. S. irio. Sonehu.s oleraceous and Vieia narhonensis to H. satirum and R. asiatieus. S. oleraceous and Mercurialis annua to R. solani. However. R. asiatieus extract was the most effective and completely inhibited growth and sporulation of the three fungi species at all incubation periods.     

77种植物提取物对植物病原菌的生长抑制作用

以稻瘟病菌(Pyricularia grisea),水稻恶苗病菌(Fusarium moniliforme),玉米大斑病菌(Exserohilum turci-cum)等12种植物病原菌为供试菌种,采用生长速率法对77种植物的95%乙醇提取物在200μg/mL下进行室内抑菌试验。结果表明有15种植物提取物对植物病原菌有抑制作用,其中华山姜、硬骨藤、龙舌兰、红蒜、大花哥纳香、海南草珊瑚对至少一种菌的抑制率在50%以上,版纳青梅、大果巴戟、华山姜等8种植物提取物对至少三种病原菌均有不同程度的抑制作用。红蒜提取物对百合炭疽病菌、海南草珊瑚提取物对番茄灰霉病菌,以及龙舌兰提取物对玉米大斑病菌的抑制率分别为61.4%、70.7%、76.6%,与阳性对照抑制率相比效果明显。     

Comparison of Arcobacter isolation methods, and diversity of Arcobacter spp. in Cheshire, United Kingdom

The aims of this study were, firstly, to compare five published methods for the isolation of Arcobacter spp. from animal feces in order to determine the most sensitive and specific method. Second, we analyzed the resulting isolates by multilocus sequence typing (MLST) in order to investigate the diversity of the isolates recovered. Third, we investigated the ability to recover Arcobacter spp. from frozen fecal samples. Seventy-seven fecal samples from cattle, sheep, and badgers were subjected to five isolation methods, based on published methods for the isolation of Arcobacter and Campylobacter spp. Thirty-nine Arcobacter butzleri isolates were analyzed using a multilocus sequence typing scheme. The survival of Arcobacter spp. in frozen samples was investigated by freezing the fecal samples at -80°C for 7 days and then applying the same five isolation methods. The most sensitive and specific method used an Arcobacter-specific broth in conjunction with modified charcoal cefoperazone deoxycholate agar (mCCDA) with added antibiotics. Freezing of fecal samples led to a reduction in the recovery of Arcobacter spp. by approximately 50%. The 39 allelic profiles obtained by MLST could be divided into 11 sequence types (STs). We have identified the most sensitive and specific method for the isolation of Arcobacter spp. from animal feces and demonstrated that the freezing of fecal samples prior to isolation reduces arcobacter recovery. MLST analysis of the isolates revealed a high level of diversity.     

Arcobacter butzleri Induce Colonic,Extra-Intestinal and Systemic Inflammatory Responses in Gnotobiotic IL-10 Deficient Mice in a Strain-Dependent Manner

Background

The immunopathological impact of human Arcobacter (A.) infections is under current debate. Episodes of gastroenteritis with abdominal pain and acute or prolonged watery diarrhea were reported for A. butzleri infected patients. Whereas adhesive, invasive and cytotoxic capacities have been described for A. butzleri in vitro, only limited information is available about the immunopathogenic potential and mechanisms of infection in vivo.

Methodology/Principal Findings

Gnotobiotic IL-10^-/- mice were generated by broad-spectrum antibiotic treatment and perorally infected with the A. butzleri strains CCUG 30485 and C1 shown to be invasive in cell culture assays. Bacterial colonization capacities, clinical conditions, intestinal, extra-intestinal and systemic immune responses were monitored at day six and 16 postinfection (p.i.). Despite stable intestinal A. butzleri colonization at high loads, gnotobiotic IL-10^-/- mice were virtually unaffected and did not display any overt symptoms at either time point. Notably, A. butzleri infection induced apoptosis of colonic epithelial cells which was paralleled by increased abundance of proliferating cells. Furthermore A. butzleri infection caused a significant increase of distinct immune cell populations such as T and B cells, regulatory T cells, macrophages and monocytes in the colon which was accompanied by elevated colonic TNF, IFN-γ, nitric oxide (NO), IL-6, IL-12p70 and MCP-1 concentrations. Strikingly, A. butzleri induced extra-intestinal and systemic immune responses as indicated by higher NO concentrations in kidney and increased TNF, IFN-γ, IL-12p70 and IL-6 levels in serum samples of infected as compared to naive mice. Overall, inflammatory responses could be observed earlier in the course of infection by the CCUG 30485 as compared to the C1 strain.

Conclusion/Significance

Peroral A. butzleri infection induced not only intestinal but also extra-intestinal and systemic immune responses in gnotobiotic IL-10^-/- mice in a strain-dependent manner. These findings point towards an immunopathogenic potential of A. butzleri in vertebrate hosts.     

The effect of citric acid, lactic acid, sodium citrate and sodium lactate, alone and in combination with nisin, on the growth of Arcobacter butzleri

The importance of Arcobacter spp. as a cause of human foodborne illness is unresolved. Organic acids and their sodium salts, and nisin are preservatives commonly used in the type of foods from which the organism is recovered. In this study their effect on the growth of A. butzleri in culture, alone and in combination, was investigated. At 0.5%, 1.0% and 2.0% lactic and citric acids inhibited A. butzleri growth; 2% sodium lactate was effective in inhibiting growth over 8 h incubation but not over longer periods. Sodium citrate was more effective than sodium lactate. Nisin alone inhibited A. butzleri growth at 500 IU ml-1 over 5 h. It did not enhance the effect of sodium citrate inhibition but it did augment the effect of sodium lactate alone over 8 h.     

The survival of three strains of Arcobacter butzleri in the presence of lemon, orange and bergamot essential oils and their components in vitro and on food

AIMS: To test the effect of oils and vapours of lemon, sweet orange and bergamot and their components against three Arcobacter butzleri strains. METHODS AND RESULTS: The disc diffusion method was used to screen the oils and vapours against three strains of A. butzleri. In vitro bergamot was the most inhibitory essential oil (EO) and both citral and linalool were effective. On cabbage leaf, the water isolate was the least susceptible to bergamot EO, citral and linalool (1-2 log reduction), with the chicken isolate being the most susceptible (6-8 log reduction). However, the latter appeared not to be susceptible to vapours over 24 h although type strain and water isolate populations reduced by 8 logs. On chicken skin, the effectiveness of the oils was reduced compared with that on cabbage leaf. CONCLUSIONS: Bergamot was the most effective of the oils tested and linalool the most effective component. All strains tested were less susceptible in food systems than in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacter isolates vary in their response to EO suggesting that the results of type strain studies should be interpreted with caution. Bergamot EO has the potential for the inhibition of this 'emerging' pathogen.     

大蒜秸秆水浸液的抑菌作用和抑茵成分初步分析

以不同浓度的大蒜秸秆水浸液对西瓜枯萎病菌、黄瓜枯萎病菌、辣椒疫霉病菌进行抑菌圈试验,并对3种不同的大蒜秸秆萃取液中的可挥发抑菌物质进行鉴定,以了解大蒜秸秆水浸液的抑菌作用。结果表明,大蒜秸秆水浸液对3种靶标菌均有抑菌活性,其最有效的抑菌浓度为100 g.L-1,对辣椒疫霉病菌的抑菌活性最强。大蒜秸秆水浸液的石油醚、三氯甲烷和乙酸乙酯3种有机溶剂萃取相对3种靶标菌均有抑菌活性,以乙酸乙酯相抑菌活性最强,在50 g.L-1浓度下对辣椒疫霉菌抑菌率达100%,在100 g.L-1浓度下对西瓜枯萎病菌和黄瓜枯萎病菌的抑菌率也达100%;三氯甲烷相的抑菌活性稍高于石油醚相;抑菌活性基本随浓度增加而增大。经GC-MS分析,初步确定大蒜秸秆水浸液中的可挥发抑菌物质主要有2-甲氧基-1,6-己二酸甲酯、邻苯二甲酸二丁酯等有机酯类,3-甲氧基-4-羟基-苯甲酸、对羟基苯甲酸等有机酸,以及对甲氧基苯酚等酚类化合物。     

Antimicrobial resistance and virulence genes profiles of Arcobacter butzleri strains isolated from back yard chickens and retail poultry meat in Chile

This research aims to investigate the presence and pathogenic potential of Arcobacter in poultry meat samples purchased in the retail market of Valdivia (South of Chile) as well as in faecal samples from backyard chickens from rural areas around this city. The isolates obtained were identified by molecular methods. Furthermore, putative virulence genes were assessed by PCR and the antimicrobial resistance was tested by phenotypic methods. Arcobacter was present in 41·6% of the samples, with the highest value in retail poultry meat (55·7%) followed by backyard production (28·0%). Arcobacter butzleri was the most prevalent species (75·6%) followed by Arcobacter skirrowii (14·8%) and Arcobacter cryaerophilus (9·6%). An 8·5% of A. butzleri strains from meat were resistant to both ciprofloxacin and tetracycline and 6·1% were resistant to erythromycin, while none was resistant to gentamycin, unlike strains from domestic chickens, which showed no resistance. Furthermore, A. butzleri strains from chicken meat presented a higher prevalence of virulence genes than strains from domestic chickens. In fact, in this last group, some genes (hecA, hecB and irgA) were completely absent. Therefore, this study provides insight on the epidemiology of Arcobacter in Chilean poultry and suggests that under traditional breeding conditions strains are, apparently, less pathogenic and drug resistant.     

中草药提取物对离体鸡小肠磷吸收和细胞活性的影响

本试验主要研究了中草药提取物对离体鸡小肠磷吸收和肠粘膜细胞活性的影响,旨在为中草药的合理选用提供参考.采用单因子完全随机设计,36只40日龄体重相近的三黄公鸡随机分成6个组,每组6个重复,每个重复1只鸡,每只鸡的十二指肠、空肠和回肠分别作为相应外翻肠囊的一个重复,测试了白头翁、马齿苋、大青叶和秦皮四味中草药的单一提取物及其组合方剂对磷吸收和肠粘膜细胞活性的影响.本试验结果表明:大青叶和秦皮对磷的吸收和肠粘膜细胞活性有负作用,白头翁、马齿苋和混合方剂对磷的吸收和肠粘膜细胞活性有正面效应.试验结论:不同中草药提取物对鸡离体小肠肠粘膜细胞活性和磷吸收有不同的影响,但具体原因还有待进一步研究.