Direct detection and identification of Arcobacter species by multiplex PCR in chicken and wastewater samples from Spain

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30 degrees C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37 degrees C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.     

石家庄市零售鸡肉样品中弓形杆菌污染调查

目的调查石家庄市零售鸡肉中弓形杆菌的污染情况,为石家庄市弓形杆菌分布特征和防控提供基础数据支持。方法从石家庄市市内四区的菜市场、超市、连锁肉店随机购买零售鸡肉样品90份,应用驱动增强动力双孔滤膜法从样品中分离培养弓形杆菌,利用弓形杆菌多重聚合酶链式反应(PCR)检测及测序的方法对分离菌株进行种属鉴定,利用16S rDNA、rpoB基因测序的方法验证菌种鉴定结果。结果 90份鸡肉样品中,60份样品检出弓形杆菌86株,其中嗜低温弓形杆菌49株、布氏弓形杆菌37株,弓形杆菌检出率为66.67%(60/90)。60份阳性样品中,嗜低温弓形杆菌阳性样品45份(75.00%),布氏弓形杆菌阳性样品35份(58.33%)。嗜低温弓形杆菌和布氏弓形杆菌混合感染阳性样品20份,占比为22.22%(20/90)。不同采样点弓形杆菌检出率有较大差异。结论石家庄市零售鸡肉中弓形杆菌污染较重,嗜低温弓形杆菌污染率高于布氏弓形杆菌,两种弓形杆菌混合污染样品率较高。     

Thermal injury and recovery of Salmonella enterica serovar enteritidis in ground chicken with temperature, pH, and sodium chloride as controlling factors

Cells of Salmonella enterica serovar Enteritidis were grown at 25 and 35 degrees C, heat injured (55, 60, and 62.5 degrees C), and recovered in tryptic soy broth (TSB) at various NaCl concentrations (2.0 and 3.5%) and pH levels (5.5 and 6.5). To assess the interactions of growth temperature, heating temperature, NaCl concentration and pH on the thermal injury and recovery of Salmonella Enteritidis in ground chicken, a randomized design with each experimental combination was used. When a logistic equation for nonlinear survival curves was used, D-values of cells of Salmonella Enteritidis grown at 25 degrees C were 7.60, 5.73, and 4.81 min at 55, 60, and 62.5 degrees C, respectively. For cells grown at 35 degrees C, the D-values were 12.38, 7.45, and 5.70 min at 55, 60, and 62.5 degrees C. The influence of tryptic soy agar and double modified lysine agar (DMLIA) on the recovery of heat-injured cells was determined. Recovery was significantly reduced on DMLIA at increased pH levels and NaCl concentrations. Higher numbers of cells were recovered in TSB with 2.0% NaCl than in TSB with 3.5% NaCl. It was observed that the rate of recovery of heat-injured cells was similar at each pH. Therefore, a pH range of 5.5 to 6.5 does not have a major inhibitory effect on the recovery of Salmonella Enteritidis.     

Detection and diversity of various Arcobacter species in Danish poultry

The prevalence and diversity of different Arcobacter spp. in various poultry species in Denmark were investigated using cultural and multiplex PCR methods. A pool of three fresh droppings obtained at the production site from 70 broiler chicken flocks aged 4-5 weeks was examined. In addition, pools of 10 cloacal swabs taken at the abattoir prior to stunning from each of 15, and 37 duck and turkey flocks, respectively, were analyzed. Thirty fresh broiler chicken carcasses and 29 cloacal swabs from the respective viscera were also examined at the abattoir. Finally, 10 caecal and 10 cloacal swabs from ducks at the abattoir were analyzed individually. In total, 85 Arcobacter isolates were obtained. Of these 45, 20 and 7 were identified as Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively, using a multiplex PCR. Interestingly, some chicken isolates of A. butzleri showed urease activity, and 6 out of seven A. skirrowi isolates were unable to hydrolyse indoxyl acetate. All chicken carcasses examined were found positive for A. butzleri and/or A. cryaerophilus, whereas 21 (72%) of the 29 chicken cloacal swabs were positive for either A. butzleri (13) or A. cryaerophilus (9). Three (4.3%) out of 70 chicken flocks analyzed were positive only for A. cryaerophilus. Of the ten ducks examined individually, 7 carried A. skirrowii and/or A. cryaerophilus in their cloacae. None of the respective caecal samples were positive. Of the remaining 15 duck flocks, 11 (73%) were positive for A. cryaerophilus (7), A. butzleri (2) or A. skirrowii (2). Four (11%) of the 37 turkey flocks analyzed harboured either A. butzleri or A. cryaerophilus. The carriage rate of Arcobacter was higher in live ducks than those of live broiler chickens and turkeys in the present study. In addition, chicken carcasses slaughtered in Denmark were found to be contaminated with Arcobacter. The presence of Arcobacter spp. both on chicken carcasses and in poultry intestine may be of significance to human health.     

PCR 法检测动物源性食品中布氏弓形菌

弓形菌是一种新型食源性致病菌,其中以布氏弓形菌污染率最高。本研究采用扩增23S rRNA的PCR法特异性检测布氏弓形菌,方法灵敏度可达103 CFU/mL。2株布氏弓形菌均特异性地扩增出了长度为2061 bp的条带;嗜低温弓形菌、斯氏弓形菌、空肠弯曲菌等共18株不同种类的菌株均无扩增产物出现,表明此PCR法能特异性的将布氏弓形菌鉴定到种一级水平。比较实验结果表明,API CAMPY鉴定试剂盒对于布氏弓形菌鉴定仅能到弓形菌属一级水平,本PCR法能实现布氏弓形菌种一级水平的鉴定,用于布氏弓形菌的检测具有优势。55份动物源性食品样品用Johnson-Murano肉汤增菌后用PCR法进行检测,其中5份样品为布氏弓形菌阳性,阳性检出率为9.1%。上述实验结果表明,本方法特异性强、操作简便,节省了检测时间,可用于动物源性食品中布氏弓形菌的快速检测。     

Inhibition of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii by plant oil aromatics

The inhibitory effect of some plant oil aromatics against three strains of Arcobacter butzleri, two strains of Arcobacter cryaerophilus, and one strain of Arcobacter skirrowii was evaluated. When MICs were determined using the broth macrodilution method, cinnamaldehyde was most inhibitory followed by thymol, carvacrol, caffeic acid, tannic acid, and eugenol (P < 0.001). Sublethal concentrations of the three most potent plant oil aromatics also were examined. Overall, cinnamaldehyde was the most bacteriostatic against all arcobacters tested except A. butzleri when these strains were exposed to the MIC25 of this aromatic aldehyde. The bacteriostatic activities of thymol and carvacrol were concentration and species dependent.     

One-step polymerase chain reaction-based typing of Arcobacter species

A species-specific PCR assay was developed for the identification of the Arcobacter species, Arcobacter butzleri, Arcobacter cryaerophilus 1A and 1B, and Arcobacter skirrowii. The primers, which amplify the most variable areas of the 23S rRNA gene, were designed to perform species-specific identification by one-step PCR. The DNA sequence of the region from A. cryaerophilus 1B was determined, and the specific pimer for the species was designed. By using one-step PCR containing the mixed primers N.butz, N.c1.A, N.c.1B, and N.ski, species-specific amplifications were detected from the reference strains of A. butzleri, A. cryaerophilus 1A, 1B, and A. skirrowii, respectively. Primers designed in this study were also evaluated on 10 of Japanese field isolates, and all species were identified. This simple one-step PCR assay was found to be a powerful tool for the survey of Arcobacter infection.     

Development of a new protocol for the isolation and quantification of Arcobacter species from poultry products.

None of the presently available selective supplements for the specific isolation of Arcobacter species allows the growth of Arcobacter butzleri, A. cryaerophilus and A. skirrowii and at the same time fully suppresses the accompanying flora present in poultry and poultry products. Furthermore, little is known about the contamination levels of poultry with Arcobacter species. In this study, a new selective supplement comprising amphotericin B (10 mg/l), cefoperazone (16 mg/l), 5-fluorouracil (100 mg/l), novobiocin (32 mg/l) and trimethoprim (64 mg/l) was developed. With a new isolation procedure, including enrichment in Arcobacter broth with the selective supplement, incubated for 24 to 48 h at 28 degrees C under microaerobic conditions, arcobacters were isolated from 100% (n = 34) of neck skin of laying hens and from 90% (n = 71) of similar samples from broilers. Of the broiler breast meat samples examined (n = 52), 65% were found to be contaminated with these bacteria. In 64% of the samples, A. butzleri was the only Arcobacter species isolated. In 9% of the samples, A. cryaerophilus was the only species present, while 11% of the samples were positive for both species simultaneously. Using direct isolation on the selective agar medium developed in this study, incubated for 24 to 48 h under microaerobic conditions at 28 degrees C. 32 out of 45 broiler carcasses and 6 out of 25 broiler breast meat samples carried a bacterial load of arcobacters of 10(2) to 10(3) cfu/g. The prevalence of Arcobacter in Belgian poultry was found higher than the prevalence of thermophilic Campylobacter species in each of the poultry categories examined. The enrichment procedure and the direct plating method were validated for the isolation of A. skirrowii. For this species, growth performance was less than the other two Arcobacter species and it was not isolated nor detected by m-PCR from the naturally contaminated poultry samples examined. This new protocol provides a fast and reliable method for the isolation of Arcobacter species from poultry and can contribute to more comprehensive epidemiological investigations.     

Thermal inactivation, growth, and survival studies of Listeria monocytogenes strains belonging to three distinct genotypic lineages

Twenty-one Listeria monocytogenes strains belonging to three different genotypic lineages were evaluated for differences between lineages and between individual strains with respect to thermal inactivation, growth, and survival. Three sets of heat inactivation conditions (60 degrees C, pH 6.0, and 0.5 M lactate; 55 degrees C, pH 6.0, and 0.5 M lactate; and 50 degrees C, pH 4.0, and 0.5 M lactate) were used on strains grown in modified brain heart infusion (BHI) broth with and without glucose. Two sets of growth conditions (35 degrees C, pH 6.5, and 0.1 M lactate and 5 degrees C, pH 6.5, and 0.1 M lactate) were used with modified BHI broths to determine lag phases and exponential growth rates. Two sets of conditions (28 degrees C, pH 4.0, and 1 M lactate and 28 degrees C, pH 4.5, and 0.5 M lactate) were used with modified BHI broth to determine survival times (D-values). Thermal inactivation D-values were consistently lowest for lineage III, but differences were not significant for any set of conditions tested. Some significant differences were observed between lineages with respect to some of the growth and survival conditions tested. Extensive strain-to-strain variation was observed for all parameters tested. Average coefficients of variation for the thermal inactivation, growth, and survival studies were 0.31, 0.18, and 0.26, respectively. Strain-to-strain variations were approximately equal to the uncertainties associated with the analytical procedures. The results obtained indicate a diversity among strains encountered in food processing that must be accounted for in process calculations and risk assessments.     

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.     

Molecular characterization of Arcobacter isolates collected in a poultry slaughterhouse

In a poultry slaughterhouse, Arcobacter contamination was examined over a period of 1 week to establish possible routes of contamination. Samples were collected from the slaughter equipment and from processing water before the onset of slaughter and from the first broiler flock slaughtered on each sampling day. Characterization of 1,079 isolates by enterobacterial repetitive intergenic consensus-polymerase chain reaction and a random amplified polymorphic DNA assay resulted in the delineation of 159 Arcobacter butzleri and 139 Arcobacter cryaerophilus types. From almost all 140 neck skin samples collected before and after evisceration, A. butzleri and A. cryaerophilus were isolated simultaneously at contamination levels ranging from 10(1) to 10(4) CFU/g. Only six A. butzleri types present in the slaughterhouse environment were also present on the broiler carcasses. None of the A. cryaerophilus genotypes were detected in both the neck skin and the environmental samples. All A. butzleri types isolated from the feather samples were also isolated from broiler neck skin samples. The slaughter equipment was contaminated with arcobacters before the onset of slaughter, but it appeared unlikely that contamination through the slaughter equipment alone explained the high contamination levels on poultry products. Arcobacters were also present in processing water, but types present in water and poultry products were different. Characterization of the Arcobacter isolates did not clarify the routes of transmission, probably because of the extreme heterogeneity among Arcobacter isolates. However, the results obtained in this study brought to light insufficient decontamination at the processing plant involved in the study and confirmed the survival capacity of certain A. butzleri strains.     

Genetic diversity of Arcobacter and Campylobacter on broiler carcasses during processing

Broiler carcasses (n=325) were sampled at three sites along the processing line (prescalding, prechilling, and postchilling) in a commercial poultry processing plant during five plant visits from August to October 2004. Pulsed-field gel electrophoresis (PFGE) was used to determine the genomic fingerprints of Camospylobacter coli (n=27), Campylobacter jejuni (n=188), Arcobacter butzleri (n=138), Arcobacter cryaerophilus 1A (n=4), and A. cryaerophilus 1B (n=31) with the restriction enzymes SmaI and KpnI for Campylobacter and Arcobacter, respectively. Campylobacter species were subtyped by the Centers for Disease Control and Prevention PulseNet 24-h standardized protocol for C. jejuni. A modification of this protocol with a different restriction endonuclease (KpnI) and different electrophoresis running conditions produced the best separation of restriction fragment patterns for Arcobacter species. Both unique and common PFGE types of Arcobacter and Campylobacter strains were identified. A total of 32.8% (57 of 174) of the Arcobacter isolates had unique PFGE profiles, whereas only 2.3% (5 of 215) of the Campylobacter isolates belonged to this category. The remaining Arcobacter strains were distributed among 25 common PFGE types; only eight common Campylobacter PFGE types were observed. Cluster analysis showed no associations among the common PFGE types for either genus. Each of the eight common Campylobacter types consisted entirely of isolates from one sampling day, whereas more than half of the common Arcobacter types contained isolates from different sampling days. Our results demonstrate far greater genetic diversity for Arcobacter than for Campylobacter and suggest that the Campylobacter types are specific to individual flocks of birds processed on each sampling day.     

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.     

Thermal resistance of spores from virulent strains of Bacillus anthracis and potential surrogates

The objective of this study was to determine the thermal resistance of spores of Bacillus anthracis and potential surrogates. The heat resistance of spores suspended in buffer (pH 7.0 or 4.5), milk, or orange juice was determined at 70, 80, and 90 degrees C. D-values for B. anthracis strains Sterne, Vollum, and Pasteur ranged from < 1 min at 90 degrees C to approximately 200 min at 70 degrees C and were lower under acidic than under neutral conditions. The D-values for B. anthracis spores fell within the range obtained for spores from eight strains of Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis. However, there were significant differences (P < 0.001) among the D-values of the strains. The z-values in pH 7.0 buffer and milk averaged approximately 10.5 degrees C and were not significantly different among strains (P < 0.05). The z-values in pH 4.5 buffer and orange juice averaged 12.9 and 13.9 degrees C, respectively, significantly (P < 0.05) higher than those obtained in milk or in pH 7.0 buffer. The significance of this difference was driven by large differences among a few strains. The z-values for B. anthracis strain Pasteur were twice as high in the acid media than in the neutral media. This study confirms that B. anthracis spores are not unusually heat resistant and that spores from validated Bacillus species are appropriate surrogates for thermal resistance studies.     

Prevalence of Arcobacter and Campylobacter on broiler carcasses during processing

Broiler carcasses (n=325) were sampled in a U.S. commercial poultry processing plant for the prevalence of Arcobacter and Campylobacter at three sites along the processing line: pre-scald, pre-chill and post-chill. Samples (75-125 broilers per site) were collected during five plant visits from August to October of 2004. Arcobacter was recovered from pre-scald carcasses more frequently (96.8%) than from pre-chill (61.3%) and post-chill carcasses (9.6%). Campylobacter was isolated from 92% of pre-scald carcasses, 100% of pre-chill carcasses, and 52% of post-chill carcasses. In total, Arcobacter was isolated from 55.1% (179 of 325), while Campylobacter was isolated from 78.5% (255 of 325) of the carcasses from the three collection sites. For Arcobacter identification, a species-specific multiplex PCR showed that A. butzleri was the most prevalent species (79.1%) followed by A. cryaerophilus 1B (18.6%). A. cryaerophilus 1A was found at low levels (2.3%). PCR identified the most common Campylobacter species as C. jejuni (87.6%) followed by C. coli (12.4%). Overall, significant contamination of broiler carcasses by Arcobacter was observed, although less than that found for Campylobacter. From pre-scald to post-chill, a far greater reduction in Arcobacter numbers was observed than for Campylobacter. Our results for Arcobacter, obtained from the same environment as the closely related pathogen Campylobacter, will aid in the development of control measures for this emerging pathogen.     

Inhibitory effect of organic acids on arcobacters in culture and their use for control of Arcobacter butzleri on chicken skin

The inhibitory effects of 17 organic acids (C₂-C₁₆ fatty acids, sorbic, benzoic, phenylacetic, fumaric, succinic, lactic, malic and citric) on Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii were investigated by determining their IC₅₀ values, defined as the concentration of acid at which the target DNA sequence was expressed at 50% of the positive control level in cultures incubated at 30 °C for 24 h. DNA was analysed by real-time PCR. The Arcobacter strains tested were inhibited by all the organic acids, with the sensitivities in the order A. skirrowii > A. cryaerophilus > A. butzleri. Eight acids with IC₅₀ values of < 1 mg/mL against A. butzleri were tested for their effects on A. butzleri inoculated on chicken carcasses at a concentration of 5 log CFU/g of skin. Inoculated halved carcasses were immersed in solutions of the acids at 5 mg/mL for 1 min. Samples of skin were collected from carcass halves after storage at 4 °C for 0, 1, 2 or 3 days for enumeration of arcobacters on Muller-Hinton agar. All eight tested acids suppressed bacterial proliferation. The highest inhibitory activities were observed for benzoic, citric, malic and sorbic acids. Subsequent sensory analysis revealed benzoic acid to be the most suitable organic acid for chicken skin treatment.     

Influence of recovery conditions on apparent heat resistance of Bacillus stearothermophilus spores

The effects of post-treatment environmental factors on the heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Nutrient Agar (NA), Antibiotic Assay Medium (AAM), Dextrose Tryptone Agar (DTA) and Tryptic Soy Agar (TSA) with Ca^{2+ added to a final concentration of 100 p.p.m. were used as recovery media. No significant differences were seen between D-values obtained except in the case of strain 12980 when comparing TSA with the other media and for strain 7953 comparing AAM and DTA. The optimum incubation temperature was slightly lower for heated than for unheated spores of} each strain, although, in general, 50 °C was adequate. Higher D-values were obtained at 50–55 °C. The effects of the pH of the medium in the range 5.0–7.0 and the addition of starch and phosphate on heat resistance have also been investigated. Maximum colony counts of heated spores were obtained at pH 7.0 and decreased as pH fell. D-values were significantly lower at pH ≤ 5.5. Increasing the concentration of phosphate in the recovery medium from 0 to 0.2% resulted in a progressive decrease in spore recovery and D-values. The addition of starch improved recoverability. The z-values obtained for the four strains studied under the different recovery conditions were similar with a mean value of 7.58 °C ± 0.28.     

OUTGROWTH OF CLOSTRIDIUM PERFRINGENS SPORES IN COOK-IN-BAG BEEF PRODUCTS1

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag ground beef that included 0.3% (w/w) sodium pyrophosphate, pH 5.5 or 7.0, and salt (sodium chloride) at 0 or 3% (w/w). The packages were processed to an internal temperature of 71.1C, ice chilled and stored at various temperatures. The total C. perfringens population was determined by plating diluted samples on tryptose-sulfite-cycloserine agar followed by anaerobic incubation for 48 h at 37C. At 28C, a combination of 3% salt and pH 5.5 was effective in delaying growth for 24 h. At 15C, growth occurred within 6 days in samples with pH 7.0, but was delayed until day 8 in the presence of 3% salt at pH 7.0. Vegetative cells were not observed even after 21 days 21 of storage at 15C in the presence 3% salt at pH 5.5. C. perfringens growth was not observed at 4C regardless of pH or salt levels. The D-values ranged from 23.33 min (3% salt, pH 7.0) to 13.99 min (3% salt, pH 5.5). Cyclic and static temperature abuse of refrigerated products for 12–15 h did not permit C. perfringens growth. However, temperature abuse of cook-in-bag beef products for periods longer than 15 h in the absence of salt led to growth of C. perfringens from a spore inoculum.     

The effect of NaCl on survival of Shigella flexneri in broth as affected by temperature and pH

Shigella, a major foodborne pathogen, survives well in salt-containing environments. However, systematic data are scarce. We studied the behavior of Shigella flexneri 5348 in brain heart infusion broth (pH 4 to 6) containing 0.5 to 8% NaCl. Stationary-phase cells were inoculated into sterile media at initial concentrations of 6 to 7 log10 CFU/ml and incubated at 12 to 37 degrees C. Bacterial population sizes were determined periodically by plate counts. Survivor curves were derived from plate count data by using a two-phase linear model to determine lag times and slopes of the curves, from which decimal reduction times (D-values) and times to a 4-log10 inactivation (T4D) were calculated. In media of pH 6, the bacteria grew in the presence of < or = 6% NaCl at 19 and 37 degrees C and in the presence of < or = 7% NaCl at 28 degrees C. In media of pH 5, growth was observed in the presence of < or = 2, < or = 4, < or = 4, and 0.5% NaCl at 37, 28, 19, and 12 degrees C, respectively. Growth did not occur and bacterial populations gradually declined in media of pH 4. While NaCl had a major effect on growth, bacterial survival was affected to a lesser extent. Lag times decreased with increasing NaCl levels; however, the effect on D-values and T4D values was less pronounced. The average T4D values for media of pH 4 containing 0.5 to 6% NaCl were 4, 13, 23, and 61 days at 37, 28, 19, and 12 degrees C, respectively. These results show that S. flexneri is salt tolerant and suggest that salty foods may serve as vehicles for infection with this bacterium.     

Thermal resistance of Salmonella enterica serotypes, Listeria monocytogenes, and Staphylococcus aureus in high solids liquid egg mixes

Decimal reduction times (D-values) were determined for Salmonella enterica serotypes, Listeria monocytogenes, and Staphylococcus aureus in two high solids egg mixes designated A and B (water activity [a(w)] = 0.76 and 0.82; solids = 53.12 and 52.63%; pH = 5.09 and 5.29; viscosity = 183 and 119 centipoise/s, respectively) using a low-volume (0.06 ml) sealed glass capillary tube procedure. For Salmonella, D-values ranged from 0.035 (70 degrees C) to 0.193 min (64 degrees C) in product A and from 0.048 to 0.193 min in product B. For Listeria, D-values ranged from 0.133 (70 degrees C) to 0.440 min (64 degrees C) in product A and from 0.074 to 0.364 min in product B. For Staphylococcus, D-values ranged from 0.332 (70 degrees C) to 1.304 min (64 degrees C) in product A and from 0.428 to 1.768 min in product B. For Listeria, the D-values of all heating temperatures were significantly higher (P < 0.01) in product A than in product B. The similar trend was also observed for Salmonella and Staphylococcus but only at 66 degrees C for Salmonella and 64 degrees C for Staphylococcus. Greater temperature dependence was observed for Salmonella inactivation in the low a(w) and low pH product (A), while the product (B) with the higher a(w) and pH had greater temperature dependence for Listeria. Compared across both egg mixes and all heating temperatures, the Staphylococcus strains were from 6.2 to 11.7 times more heat resistant than S. enterica serotypes and from 2.2 to 7.5 times more heat resistant than L. monocytogenes.