Sensitivity of three methods used in the isolation of Arcobacter spp. in raw ground pork

Arcobacter, an aerotolerant Campylobacter-like organism, has been designated an emerging pathogen because of its newly recognized ability to cause diarrheal illness in both humans and animals and its presence in the human food supply. Because there is no standard isolation method for its detection, the true occurrence of this pathogen is largely unknown. In addition, the lack of a standardized isolation protocol limits the ability of investigators to compare field data. Arcobacter has been detected in whole muscle and ground pork at various levels by two different isolation methods (those of deBoer and Collins). In this study, these methods were tested along with the Johnson-Murano (JM) method, developed in our laboratory. The sensitivity of each method was tested for ground pork inoculated with Arcobacter butzleri and Arcobacter cryaerophilus IA at levels of 10(4), 10(3), 10(2), and 10(1) CFU/g. Controls included tubes with uninoculated pork and broth tubes without pork. All samples that were morphologically similar to Arcobacter were analyzed by Gram staining and by catalase and oxidasereactions. Presumptive positive samples were confirmed by the polymerase chain reaction. The JM method was determined to be the most sensitive, detecting A. butzleri down to a level of 10(1) CFU/g in 100% of the samples and detecting A. cryaerophilus IA at a level of 10(1) CFU/g in 75% of samples. In a pure buffer system, the Collins method was as effective as the JM method in isolating both organisms to levels of 10(1) cells per g.     

A comparison of three methods for the isolation of Arcobacter spp. from retail raw poultry in Northern Ireland

Recent evidence suggests that arcobacters, especially Arcobacter butzleri, are potential foodborne pathogens, but standardized detection methods have yet to be established. A study was undertaken to determine which of three isolation methods was the most effective for the isolation of Arcobacter spp. from fresh raw poultry. Methods 1 was microaerobic and involved a membrane filtration step followed by plating onto blood agar. Method 2 was also microaerobic and involved enrichment and plating media containing a five-antibiotic cocktail. Method 3 was aerobic and was based on enrichment in a charcoal-based broth containing two antibiotics. Retail poultry samples (n = 50) were obtained from supermarkets in Northern Ireland; the European Community license number was recorded to ensure sample diversity. Presumptive arcobacters were identified using genus-specific and species-specific primers. Methods 1 resulted in the lowest recovery of arcobacters (28% of samples positive). The detection rate for method 2 (68%) was higher than that for method 3 (50%), but the difference was not significant (P > 0.05). Modification of method 3 by plating the enrichment broth at 24 h, as well as at 48 h, increased recovery to 68%. Use of methods 2 and 3 together increased the number of positive samples detected by approximately 25% compared with use of either method alone. A. butzleri was the most commonly isolated species using all methods. Method 3 detected Arcobacter cryaerophilus in more samples (n = 3) than did method 1 and 2 (n = 1). Arcobacter skirrowii was detected by only method 3 (n = 1). In terms of sensitivity, ease of use, and diversity of species recovered, modified method 3 was the overall method of choice.     

Prevalence of Arcobacter spp. in raw milk and retail raw meats in Northern Ireland

A 1-year study was undertaken to determine the prevalence of Arcobacter spp. in raw milk and retail raw meats on sale in Northern Ireland. Retail raw poultry samples (n = 94), pork samples (n = 101), and beef samples (n = 108) were obtained from supermarkets in Northern Ireland, and raw milk samples (n = 101) were kindly provided by the Milk Research Laboratory, Department of Agriculture and Rural Development, Belfast, Northern Ireland. Presumptive arcobacters were identified by previously described genus-specific and species-specific PCR assays. Arcobacter spp. were found to be common contaminants of retail raw meats and raw milk in Northern Ireland. Poultry meat (62%) had the highest prevalence, but frequent isolations were made from pork (35%), beef (34%), and raw milk (46%). Arcobacter butzleri was the predominant species isolated from retail raw meats and was the only species isolated from raw milk samples. Arcobacter cryaerophilus was detected less frequently, and Arcobacter skirrowii was detected only as a cocontaminant. To our knowledge, this is the first report of Arcobacter spp. prevalence in a diverse range of products of animal origin in Northern Ireland.     

Prevalence and diversity of Arcobacter spp. in the Czech Republic

The aim of this study was to examine 634 samples of chicken, lamb, pork, beef, fish, samples from the intensive animal industry and from poultry for slaughter, as well as from the domestic breeding of poultry, horses, pigs, and lambs, from surface water, and from clinical samples for the presence of Arcobacter. All the samples were examined with a cultivation method, followed by confirmation by multiplex PCR. The method of multiplex PCR applied directly to a liquid medium after enrichment was applied only to the samples with the highest probability of the presence of arcobacters. Arcobacter spp. were detected in 11.8% of the samples, of which A. butzleri, A. cryaerophilus, and A. skirrowii were found in 6.6, 5.1, and 0.2% of the samples, respectively. The sources of the arcobacters were chicken meat from the retail market, intensive animal production facilities, domestic chicken breeding facilities, lamb raising environments, surface water and wastewater, and beef swabs taken in a meat processing factory. No occurrence of arcobacters was identified in the swabs from slaughter turkeys, ducks, and wild poultry. No arcobacters were found in horse and pig breeding environments, on pork, or on the swabs of fish. Forty-two rectal swabs taken from humans were also free of Arcobacter. Seventeen isolates of Arcobacter were further identified by sequencing the 16S rRNA gene. Varied genotypes were observed among A. butzleri from chicken meat and chicken breeds, and A. cryaerophilus from wastewater and chicken breeds. They were similar to the genotypes present in wastewater, porcine feces, human stool, and human blood obtained from databases. Our results revealed that the chicken meat from the retail market is an important source of arcobacters. Cross-contamination during handling of chicken carcass practices could play a key role in the spread of Arcobacter.     

Prevalence and Concentration of Arcobacter spp. on Australian Beef Carcasses

The International Commission on Microbiological Specifications for Foods (ICMSF) classified Arcobacter spp. as emerging pathogens in 2002. Arcobacter spp. have been isolated from numerous food products at retail and from animal carcasses and feces at slaughter. A survey was conducted to determine both the prevalence and concentration of Arcobacter spp. on prechill beef carcasses. Surface swab samples were collected from 130 beef carcasses at the end of processing, prior to chilling. The concentration of Arcobacter spp. was determined by a most-probable-number per square centimeter (3 by 3) method with a limit of detection of 0.12 CFU/cm(2). Of the 100 carcasses examined from export abattoirs, 20 (20.0%) were contaminated with Arcobacter spp., and 5 of these had quantifiable levels of contamination ranging from 0.12 to 0.31 CFU/cm(2). Of the 30 carcasses examined at a pet food abattoir, 25 (83.3%) were contaminated with Arcobacter spp., and 10 of these had quantifiable levels of contamination ranging from 0.12 to 0.95 CFU/cm(2). Three species of Arcobacter, A. butzleri, A. cryaerophilus, and A. skirowii, were identified by PCR. Each of the species was present in an approximately equal ratio from export abattoirs. This study demonstrates that slaughter practices at export abattoirs are sufficient to maintain both low prevalence and low levels of contamination of beef carcasses with Arcobacter spp.     

Development of a new medium for the isolation of Arcobacter spp.

Arcobacter, the newly reclassified Campylobacter species, has been shown to cause diarrhea in both humans and animals. Few studies have been conducted regarding its occurrence in foods because of the lack of effective isolation and identification methods. The purpose of this study was to develop a plating medium that would be selective for the three most commonly found Arcobacter species. The effect of common components used in media intended for the isolation of Campylobacter, Helicobacter, and other gram-negative rods was examined. These components were divided into five distinct groups: (1) basic growth nutrients, (2) reducing and growth-promoting agents, (3) detoxifying agents, (4) antibiotics, and (5) color-enhancing compounds. Components from each of these groups were tested for their ability to recover Arcobacter on a solid medium when incubated aerobically at 30 degrees C for up to 72 h. Growth was evaluated by the ecometric technique, colony size, and differential colony morphology after incubation. After initial evaluations, five formulas showing the best results were selected and tested in detail and compared with brucella agar. A medium containing a basal nutrient mix along with 0.05% thioglycolic acid, 0.05% sodium pyruvate, and 5% sheep's blood (pH 6.9+/-0.2) was found to be the most effective for the growth of A. butzleri, A. cryaerophilus, and A. nitrofigilis. In addition to superior growth characteristics, a deep red color around the colonies also was observed with this formulation.     

Determination of the occurrence of Arcobacter butzleri in beef and dairy cattle from Texas by various isolation methods

Arcobacter butzleri is a pathogenic bacterium that has been found in dairy cattle, pigs, poultry, and humans. As of this writing, there are no data on the incidence of A. butzleri in beef cattle. Given the differences in rearing practices used for feedlot cattle and those used for dairy cattle, differences in the incidences of this organism in various types of cattle may also exist. Numerous culture methods have been used to isolate A. butzleri, but there are few data on the comparative efficacies of these methods. The objectives of this study were to determine the incidence of A. butzleri in cattle from Texas and to compare the effectiveness levels of the Johnson-Murano (JM) method (consisting of enrichment in JM broth followed by plating on JM agar) and the Collins method (consisting of enrichment in EMJH-P80 broth followed by plating on Cephalothin, Vancomycin, and Amphotericin B [CVA] agar) in the isolation of this organism. Fifty cattle each from two feedlots, a dairy, and a stocker yard were sampled. Fecal swabs were obtained from cattle, and each sample was cultured by the JM method, the Collins method, and combinations of the two methods with the broth of one method being used with the agar of the other. Polymerase chain reaction was used to identify the isolates for confirmation of A. butzleri. Samples from 18 of 200 cattle tested positive for A. butzleri. This organism was detected by the JM method in 4.5% of the samples and by the Collins method in 2.5% of the samples. An incidence of 4.0% was found when JM broth was used with CVA agar, while no samples tested positive for A. butzleri when EMJH-P80 broth was used with JM agar.     

A comparison of media for the isolation of Arcobacter spp. from retail packs of beef

In order to determine the most effective protocol for the isolation of Arcobacter spp. from retail packs of beef, three published methods (A, B, and C) were selected. In addition, a modified version of method B was studied (method D). The ability of the four methods to isolate Arcobacter from standardized beef samples (n = 80) was compared with presumptive Arcobacter isolates being identified to genus and species level, using multiplex PCR methods. The presence of Arcobacter in enrichment broths was also investigated using PCR techniques. Overall, the modified enrichment and selection media of Johnson and Murano (method D) gave the highest recovery of Arcobacter. Recovery using these media was enhanced by incubating the enrichment and selection media in a microaerobic cabinet rather than air, and the inclusion of streaking the enrichment broth onto selective agar after 24 h in addition to 48 h. Method D yielded significantly more Arcobacter-positive samples of beef (P < 0.01) than did the three other methods investigated.     

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid (YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm(2). This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.     

Prevalence and Distribution of Arcobacter spp. in Raw Milk and Retail Raw Beef

A total of 106 beef samples which consisted of local (n = 59) and imported (n = 47) beef and 180 milk samples from cows (n = 86) and goats (n = 94) were collected from Selangor, Malaysia. Overall, 30.2% (32 of 106) of beef samples were found positive for Arcobacter species. Imported beef was significantly more contaminated (46.80%) than local beef (16.9%). Arcobacter butzleri was the species isolated most frequently from imported (81.8%) and local (60%) beef, followed by Arcobacter cryaerophilus in local (33.3%) and imported (18.2%) beef samples. Only one local beef sample (10%) yielded Arcobacter skirrowii. Arcobacter species were detected from cow's milk (5.8%), with A. butzleri as the dominant species (60%), followed by A. cryaerophilus (40%), whereas none of the goat's milk samples were found positive for Arcobacter. This is the first report of the detection of Arcobacter in milk and beef in Malaysia.     

Prevalence and numbers of Salmonella and Campylobacter spp. on raw,whole chickens in relation to sampling methods

Salmonella and Campylobacter continue to be major foodborne pathogens and raw poultry is considered to be an important source of these bacteria. In this study, the prevalence and numbers of Salmonella and Campylobacter spp. in relation to isolation/sampling methods were determined in 241 whole raw chickens purchased from retail outlets in England during the winters of 1998/1999 (101 chickens) and 1999/2000 (140 chickens). The packaging of the 140 chickens was also examined for the presence of the above pathogens. The prevalence and numbers of enterococci were examined in 21 of the 101 chickens. In total, Salmonella and Campylobacter spp. were present in 25% and 83% of the chickens, respectively. Salmonella were isolated from a sample representing both the inside and outside of the packaging in 19% of the chickens, while the corresponding figure for Campylobacter spp. was 56%. Both of these pathogens were isolated from the outside of the packaging in 6% of the chickens. Salmonella was more frequently isolated from samples containing chicken skin in comparison with those containing carcass-rinse fluid only. Two chickens (0.8%) were positive for Salmonella by direct enumeration methods with contamination levels of log10 3.8 and 4.5 colony forming units (cfu) per carcass, respectively. The most prevalent serotypes were S. Hadar, S. Enteritidis and S. Indiana and two different serotypes were identified in 5/20 salmonella-positive chickens. Resistance to at least one antibiotic was found in 70% of the strains, 46% were multiresistant (resistant to > or = four drugs) and 52% showed a lowered susceptibility to ciprofloxacin. The likelihood of isolating Campylobacter spp. from neck-skin, carcass-rinse or carcass-rinse plus whole skin samples was similar, Campylobacter spp. were found in higher levels in carcass-rinse or carcass-rinse plus whole skin samples than in neck-skin. The log10 cfu of Campylobacter spp. were 2.70-4.99 in 18% of the chickens and 5.00-6.99 in 20%. Campylobacter isolates (425) comprised Campylobacter jejuni (98%) and C. coli (2%) and 98 different sero/phagetypes of these two species were identified. Resistance to at least one antibiotic was found in 73% of the strains and 13% were multiresistant. Thirteen percent of the strains showed lowered susceptibility to ciprofloxacin, while 4.9% were resistant to erythromycin. Vancomycin-resistant enterococci (VRE), able to grow on agar containing 15 mg l(-1) vancomycin (VRE15), were present in 19 chickens. The log10 cfu of VRE15 was 2.90-3.99 in 10 chickens and between 4.00 and 4.99 in two chickens. The data presented here contribute to risk assessment and highlight the need to continue to emphasise the safe handling of raw retail poultry.     

北京市集贸市场生鲜猪肉肠球菌的耐药特征分析

目的了解北京市某大型集贸市场内部环境样品和零售生鲜猪肉中分离肠球菌的耐药性特征。方法采用肉汤稀释法测定86株肠球菌对10种临床常用抗生素的药物敏感性。结果 86株肠球菌对四环素、红霉素、高浓度链霉素、环丙沙星、高浓度庆大霉素和氯霉素的耐药率分别为67.44%(58/86)、54.65%(47/86)、38.37%(33/86)、26.74%(23/86)、23.26%(20/86)和20.93%(18/86),耐3种以上抗生素的多重耐药肠球菌有30株,占34.88%,耐药谱共有22种。所有肠球菌对氨苄西林、青霉素和万古霉素均敏感。耐药谱型分析发现,不同样品来源肠球菌的耐药谱差异较大。另外,本研究从国内食品中分离到对达托霉素耐药的肠球菌,此前我国未见相关报道。结论北京市集贸市场内部环境和零售生鲜猪肉中分离的肠球菌耐药严重,并呈现一定的耐药谱型差异,而且分离到达托霉素耐药株,应引起关注。     

Dynamic model for predicting growth of Salmonella spp. in ground sterile pork

A predictive model for Salmonella spp. growth in ground pork was developed and validated using kinetic growth data. Salmonella spp. kinetic growth data in ground pork were collected at several isothermal conditions (between 10 and 45 °C) and Baranyi model was fitted to describe the growth at each temperature, separately. The maximum growth rates (μ_max) estimated from the Baranyi model were modeled as a function of temperature using a modified Ratkowsky equation. To estimate bacterial growth under dynamic temperature conditions, the differential form of the Baranyi model, in combination with the modified Ratkowsky equation for rate constants, was solved numerically using fourth order Runge-Kutta method. The dynamic model was validated using five different dynamic temperature profiles (linear cooling, exponential cooling, linear heating, exponential heating, and sinusoidal). Performance measures, root mean squared error, accuracy factor, and bias factor were used to evaluate the model performance, and were observed to be satisfactory. The dynamic model can estimate the growth of Salmonella spp. in pork within a 0.5 log accuracy under both linear and exponential cooling profiles, although the model may overestimate or underestimate at some data points, which were generally < 1 log. Under sinusoidal temperature profiles, the estimates from the dynamic model were also within 0.5 log of the observed values. However, underestimation could occur if the bacteria were exposed to temperatures below the minimum growth temperature of Salmonella spp., since low temperature conditions could alter the cell physiology. To obtain an accurate estimate of Salmonella spp. growth using the models reported in this work, it is suggested that the models be used at temperatures above 7 °C, the minimum growth temperature for Salmonella spp. in pork.     

Prevalence of Clostridium difficile isolated from various raw meats in Korea

Food Science and Biotechnology - Clostridium difficile is an anaerobic, toxin-producing pathogen that causes human infection possibly through the consumption of meat. Clostridium difficile was...     

The introduction of Arcobacter spp. in poultry slaughterhouses

Despite the presence high levels of Arcobacter spp. on chicken carcasses, the source of arcobacter contamination in slaughterhouses still remains unclear. It has been hypothesised in the literature that Arcobacter species that contaminate carcasses originate in in-plant slaughterhouses and/or supply water. The present study aimed to determine the source of Arcobacter contamination in two poultry slaughterhouses in The Netherlands. Carcasses and intestinal tracts from 3 hen flocks and 2 broiler flocks were collected. Water draining off carcasses during processing in 2 slaughterhouses and supply water in one slaughterhouse were also taken. For one flock, cloacal swabs and faecal samples were taken on the farm before slaughtering. ERIC-PCR was applied to study the genetic diversity and relationship among the isolates. No Arcobacter spp. were found in the supply water but on almost all of the sampled carcasses and in carcass-draining-off water arcobacters were identified. Arcobacter spp. were detected in the gut systems of chickens, ranging from 20% to 85% in hens and 3.3% and 51% in broilers. Similar ERIC-PCR genotypes were detected in gut contents as well as on carcasses from the same flock. The present study demonstrated that Arcobacter spp. can be detected in chicken intestines at slaughter and could be brought in this way into slaughterhouses where the bacteria contaminate carcasses during processing.     

Study of Arcobacter spp. contamination in fresh lettuces detected by different cultural and molecular methods

Arcobacters are considered potential emerging food and waterborne pathogens. However, there is no data on the presence of Arcobacter spp. in fresh vegetables. Therefore the objective of this research was to study the presence of Arcobacter spp. in fresh lettuces.Fifty fresh lettuces purchased from different local shops in Valencia (Spain) were analyzed. The assay was performed simultaneously by cultural and molecular methods. Isolates were identified by real-time, multiplex PCR and restriction fragment length polymorphism analysis of PCR-amplified DNA fragment (PCR-RFLP). Finally, all the isolates were genotyped using the randomly amplified polymorphic DNA (RAPD-PCR) method.Arcobacter sp. was detected in 10 of the 50 samples (20%) by real-time PCR, being A. butzleri the unique detected species by mPCR. The detection levels obtained by conventional PCR (7 samples/50, 14%) were slightly lower. These seven samples were found to be positive also by culture isolation. All 19 obtained isolates were identified as A. butzleri by multiplex PCR and PCR-RFLP. Great genetic heterogeneity among the isolates was observed by RAPD-PCR profiling.To our knowledge, this is the first study in which Arcobacter spp. is detected in fresh vegetables such as lettuces. Although these foods are generally considered safe, given the large quantities consumed and the fact that further cooking is absent, lettuce could be a source of Arcobacters of public health concern.     

Comparison of three protocols for the isolation of Arcobacter from poultry.

The microaerophilic bacterium Arcobacter has received increasing attention in recent years regarding its presence in food products. There exist a limited number of methods for the detection of this microorganism, with currently available methods being cumbersome to perform, time consuming, and limited in specificity. The objective of this study was to develop a selective enrichment broth to isolate accurately three Arcobacter spp. from concentrated chicken microflora by comparing the efficacy of various selective and growth-promoting additives in order. This newly developed enrichment broth was incorporated into an isolation protocol using a previously developed plating medium, and this new protocol was compared with two existing methods for the isolation of Arcobacter from poultry. Method 1 consisted of enrichment in Ellinghausen-McCullough-Johnson-Harris Polysorbate 80 broth followed by plating on cefoperazone-vancomycin-amphotericin B medium. Method 2 consisted of enrichment in Arcobacter selective broth and plating onto Arcobacter selective medium. Method 3 (the JM method), used a newly developed enrichment broth followed by plating on a previously described JM agar. The JM method isolated Arcobacter strains in 42 out of 50 broiler chicken samples, while methods 1 and 2 detected the organism in only 24 and 15 out of 50 samples, respectively.     

Antioxidative properties of onion peel extracts against lipid oxidation in raw ground pork

Onion peels extracted with various concentrations of ethanol were examined for their extraction yield, total phenolic content, DPPH scavenging activity, and reducing power. Onion peel extracted with 50% ethanol exhibited higher extraction yields, total phenolic content, and DPPH scavenging activity than extracts from distilled water, 70 or 95% ethanol. Thus, the antioxidative efficiency to inhibit lipid oxidation of ascorbic acid (0.05%) and 50% ethanol extract of onion peel (0.05, 0.1, and 0.2%) in raw ground pork was investigated during chilled storage (16 days). The pH of the samples decreased considerably during storage (p<0.05). Addition of ethanol onion peel extract significantly improved the thiobarbituric acid reactive substances and peroxide values in raw ground pork meat (p<0.05). Therefore, onion peel extract by 50% ethanol is a very effective inhibitor of lipid oxidation and has potential as a natural antioxidant in raw ground pork.     

食品及环境样品中肠球菌快速检验方法的 建立及优化

目的建立并优化食品及环境样品中肠球菌的快速检验方法。方法用mEI培养基作为选择性培养基,肠球菌tuf基因为靶基因设计引物,建立食品及环境样品中肠球菌的快速检验方法。通过添加肠球菌和非肠球菌标准菌株,验证方法的检测限、灵敏度及特异性,并使用优化后的方法对北京市丰台区某大型生鲜猪肉集贸市场的地面、墙壁、污水和生鲜猪肉等样品中的肠球菌进行检验。结果所建方法的检测限为1 CFU/100cm2和1 CFU/25 g(mL),且与10种非肠球菌无交叉反应;地面涂抹、墙壁涂抹、污水和生鲜猪肉等样品中均检验出肠球菌,检出率为100%。结论建立了简便、快速、灵敏、特异的肠球菌检验方法;集贸市场内部环境和生鲜猪肉存在肠球菌污染。应加强市售生鲜肉中肠球菌的监测,并采取相应措施降低其污染水平。