Inhibitory Effects of Carrageenans on Endotoxin-Induced Inflammation.

The inhibitory effects of carrageenans (CRGs) on lipopolysaccharide (LPS) induced inflammation in a mouse model of endotoxemia and in complex therapy of patients with enteric infections of Salmonella etiology were studied. The atomic force microscopy (AFM) examination of LPS and its mixture with CRGs showed that the LPS morphology is significantly changed under the action of κ- and κ/ß-CRGs. CRGs were able to increase the synthesis of anti-inflammatory interleukin 10 (IL-10) in vitro, and, at low concentrations, their activity in the mixture with LPS was higher. The protective effect of CRGs against Escherichia coli LPS was studied in vivo by monitoring the biochemical and pathomorphological parameters. The κ- and κ/ß-CRGs and food supplement "Carrageenan-FE" increased the nonspecific resistance of mice to E. coli LPS at the expense of the inhibition of processes of thymus involution, adrenals hypertrophy, thyroid atrophy, hypercorticoidism, glycogenolysis, and lactate acidosis. The estimation of the therapeutic action of food supplement Carrageenan-FE in complex therapy of patients with enteric infections of Salmonella etiology is given. Carrageenan-FE restores the system of hemostasis and corrects some biochemical indicators and parameters in the immune systems of patients. These results allow us to hope for the practical application of CRGs for lowering the endotoxemia level in patients under the development of the infectious process caused by Gram-negative bacteria.

Transcriptional profiling of Salmonella enterica serovar Enteritidis exposed to ethanolic extract of organic cranberry pomace.

Non-typhoidal Salmonella enterica serovars continue to be an important food safety issue worldwide. Cranberry (Vaccinium macrocarpon Ait) fruits possess antimicrobial properties due to their various acids and phenolic compounds; however, the underlying mechanism of actions is poorly understood. We evaluated the effects of cranberry extracts on the growth rate of Salmonella enterica serovars Typhimurium, Enteritidis and Heidelberg and on the transcriptomic profile of Salmonella Enteritidis to gain insight into phenotypic and transcriptional changes induced by cranberry extracts on this pathogen. An ethanolic extract from cranberry pomaces (KCOH) and two of its sub-fractions, anthocyanins (CRFa20) and non-anthocyanin polyphenols (CRFp85), were used. The minimum inhibitory (MICs) and bactericidal (MBCs) concentrations of these fractions against tested pathogens were obtained using the broth micro-dilution method according to the Clinical Laboratory Standard Institute's guidelines. Transcriptional profiles of S. Enteritidis grown in cation-adjusted Mueller-Hinton broth supplemented with or without 2 or 4 mg/ml of KCOH were compared by RNASeq to reveal gene modulations serving as markers for biological activity. The MIC and MBC values of KCOH were 8 and 16 mg/mL, respectively, against all tested S. enterica isolates. The MIC value was 4 mg/mL for both CRFa20 and CRFp85 sub-fractions, and a reduced MBC value was obtained for CRFp85 (4 mg/ml). Treatment of S. Enteritidis with KCOH revealed a concentration-dependent transcriptional signature. Compared to the control, 2 mg/ml of KCOH exposure resulted in 89 differentially expressed genes (DEGs), of which 53 and 36 were downregulated and upregulated, respectively. The upregulated genes included those involved in citrate metabolism, enterobactin synthesis and transport, and virulence. Exposure to 4 mg/ml KCOH led to the modulated expression of 376 genes, of which 233 were downregulated and 143 upregulated, which is 4.2 times more DEGs than from exposure to 2 mg/ml KCOH. The downregulated genes were related to flagellar motility, Salmonella Pathogenicity Island-1 (SPI-1), cell wall/membrane biogenesis, and transcription. Moreover, genes involved in energy production and conversion, carbohydrate transport and metabolism, and coenzyme transport and metabolism were upregulated during exposure to 4 mg/ml KCOH. Overall, 57 genes were differentially expressed (48 downregulated and 9 upregulated) in response to both concentrations. Both concentrations of KCOH downregulated expression of hilA, which is a major SPI-1 transcriptional regulator. This study provides information on the response of Salmonella exposed to cranberry extracts, which could be used in the control of this important foodborne pathogen.

Efficacy of plant-derived antimicrobials for controlling Salmonella Schwarzengrund on dry pet food.

Salmonella enterica is a major human pathogen that is responsible for 23,000 hospitalizations annually in the United States. Contact with contaminated pet food and infected companion animals can transmit salmonellosis to humans. Recent multistate human outbreaks of salmonellosis linked to commercial contaminated dry dog foods underscore the need for controlling the pathogen in pet foods for protecting pet and public health. In this study, the efficacy of five Generally Recognized as Safe (GRAS) status, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde (TC), carvacrol (CR), thymol (TY), eugenol (EG), and caprylic acid (CA) applied as a vegetable oil or chitosan based antimicrobial spray on dry pet food for reducing Salmonella Schwarzengrund was investigated. Three hundred gram portions of a commercial dry dog food were inoculated with a two-strain mixture of nalidixic acid (NA) resistant S. Schwarzengrund (~6 log CFU/g), followed by a spray treatment with 0%, 0.5%, 1% or 2% of TC, CR, TY, EG or CA in combination with 5% vegetable oil or 1% chitosan as a carrier. The control and treated dog food samples were stored at 25 °C for 28 days. On days 0, 1, 3, 5, 7, 14, 21, and 28, Salmonella on pet food was enumerated by serial dilution and plating on xylose lysine desoxycholate (XLD) agar. All PDAs at 1% and 2% applied in vegetable oil or chitosan reduced S. Schwarzengrund by at least ~2 log CFU/g on day 3 of storage when compared to control (P < 0.05). No significant reductions in Salmonella were observed on feed sprayed with only vegetable oil or chitosan (P > 0.05). Overall, 2% TC in vegetable oil or chitosan was the most effective treatment, where at least 3 to 3.5 log CFU/g reduction in bacterial populations was observed during storage (P < 0.05). Results suggest that the aforementioned PDAs could potentially be used as an antimicrobial spray to reduce S. Schwarzengrund on dry dog food. However, further studies on the acceptance of PDA-treated dry food by dogs are needed.

Tolerance to benzalkonium chloride and antimicrobial activity of Butia odorata Barb. Rodr. extract in Salmonella spp. isolates from food and food environments.

Salmonellosis, caused by the consumption of contaminated foods, is a major health problem worldwide. The aims of this study were to assess the susceptibility of Salmonella spp. isolates to benzalkonium chloride (BC) disinfectant and the antimicrobial activity of Butia odorata Barb. Rodr. extract against the same isolates from food and food environments. Moreover, phenotypic and genotypic resistance profiles, the presence of virulence genes and biofilm forming ability were determined. The minimum inhibitory concentration (MIC) of B. odorata extract against Salmonella spp. ranged from 10 to >19 mg.mL-1. Resistance to ampicillin, streptomycin, nalidixic acid, sulfonamide, trimethoprim/sulfamethoxazole, trimethoprim, tetracycline, and chloramphenicol was observed. In addition, multidrug resistance was observed in seven isolates (26.92%). The MIC of BC ranged from 32 to 64 mg.L-1, higher concentrations in comparison with wild-type MICs, and therefore were considered tolerant. Several resistance genes were detected, of which the most common were aadA, qacEΔ1, blaTEM, int1, sul1, and tetA. All isolates carried at least one virulence gene and produced biofilms on stainless steel surfaces at 10 and 22 °C. On the other hand, the B. odorata extract showed activity against Salmonella spp., and it has the potential to be used as a natural antimicrobial to control this important foodborne pathogen, despite its virulence potential and antimicrobial resistance profile.

In-package atmospheric cold plasma treatment of bulk grape tomatoes for microbiological safety and preservation.

Effects of dielectric barrier discharge atmospheric cold plasma (DACP) treatment on the inactivation of Salmonella and the storability of grape tomato were investigated. Grape tomatoes, with or without inoculation with Salmonella, were packaged in a polyethylene terephthalate (PET) commercial clamshell container and cold plasma-treated at 35 kV at 1.1 A for 3 min using a DACP system equipped with a pin-type high-voltage electrode. DACP treatment inactivated Salmonella (p < 0.05) without altering the color or firmness of the grape tomatoes (p > 0.05). DACP treatment inactivated Salmonella uniformly in both layers of the double-layer configuration of the grape tomatoes regardless of the position of the tomatoes in each layer. Salmonella was most efficiently inactivated when the headspace to tomato volume ratio of the container was highest. Integration of rolling of tomatoes during treatment significantly increased the Salmonella reduction rates from 0.9 ±â€¯0.2 log CFU/tomato to 3.3 ±â€¯0.5 log CFU/tomato in the double-layer configuration of the tomato samples. Rolling-integrated DACP also initially reduced the number of total mesophilic aerobic bacteria and yeast and molds in the double-layer configuration of tomato samples by 1.3 ±â€¯0.3 and 1.5 ±â€¯0.2 log CFU/tomato, respectively. DACP treatment effectively reduced the growth of Salmonella and indigenous microorganisms at 10 and 25 °C, and did not influence the surface color, firmness, weight loss, lycopene concentration and residual ascorbic acid of grape tomatoes during storage at 10 and 25 °C. DACP treatment holds promise as a post-packaging process for improving microbial safety against Salmonella and storability of fresh grape tomatoes.

Citrus fruit extracts with carvacrol and thymol eliminated 7-log acid-adapted Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes: A potential of effective natural antibacterial agents.

Despite the widespread belief that citrus fruit extracts (CFEs) are microbiologically safe due to their acidity, limited bactericidal effect results in low applicability as antibacterial agent and outbreaks occurred by acid-adapted pathogens. Here, we examined the antibacterial effects of CFEs [lime (Citrus medica), lemon (Citrus limon), calamansi (Citrus microcarpa)] combined with essential oil components (EOCs; carvacrol and thymol) against non-acid-adapted/acid-adapted Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes under 22 °C for 5 min. CFEs (<20%) alone or small amounts of EOCs (2.0 mM; 0.032%) alone could not inactivate the target bacteria effectively. However, combined treatments exhibited marked synergy: CFE + EOCs eliminated all the bacteria (>6.9 log CFU/ml). Among the CFEs tested, the highest synergism was shown by calamansi, an exotic citrus fruit previously unrecognized as an antibacterial agent. Although acid-adaptation improved bacterial survival, calamansi (<20%) + EOCs (<0.032%) completely inactivated even the most resistant pathogen (E. coli O157:H7). Validation test also showed that all tested commercial juice products also eliminated acid-adapted pathogens when used with EOCs. Physicochemical analysis of tested CFEs (pH measurement and HPLC analysis of components) revealed that low pH and flavanone (hesperidin) did not contribute to the synergistic bactericidal effects. Rather, the high citric acid content is likely to contribute to the strong synergistic effect with EOCs by damaging susceptible bacterial membranes. Sensory scores for CFEs were not altered by addition of EOCs at concentrations up to 1.5 mM. This study provides new insight into the utility of CFEs with EOCs to improve not only the microbiological safety of food products containing CFEs but also their applicability as natural antibacterial complex.

Modeling Growth Kinetic Parameters of Salmonella Enteritidis SE86 on Homemade Mayonnaise Under Isothermal and Nonisothermal Conditions.

During the last decade, a specific strain of Salmonella Enteritidis (named SE86) has been identified as the major etiological agent responsible for salmonellosis in the State of Rio Grande do Sul, Southern Brazil, and the main food vehicle was homemade mayonnaise (HM). This study aimed to model the growth prediction of SE86 on HM under isothermal and nonisothermal conditions. SE86 was inoculated on HM and stored at 7, 10, 15, 20, 25, 30, and 37°C. Growth curves were built by fitting data to the Baranyi's DMFit, generating r(2) values greater than 0.98 for primary models. Secondary model was fitted with Ratkowsky equation, generating r(2) and root mean square error values of 0.99 and 0.016, respectively. Also, the growth of SE86 under nonisothermal conditions simulating abuse temperature during preparation, storage, and serving of HM was studied. Experimental data showed that SE86 did not grow on HM at 7°C for 30 days. At 10°C, no growth was observed until approximately 18 h, and the infective dose (assumed as 10(6) CFU/g) was reached after 8.1 days. However, the same numbers of SE86 were attained after 6 hours at 37°C. Experimental data demonstrated shorter lag times than those generated by ComBase Predictive Models, suggesting that SE86 is very well adapted for growing on HM. SE86 stored under nonisothermal conditions increased population to reach about 10(6) CFU/g after approximately 30 hours of storage. In conclusion, the developed model can be used to predict the growth of SE86 on HM under various temperatures, and considering this pathogen, HM can be produced if safe eggs are used and HM is stored below 7°C.

Travel-associated antimicrobial drug-resistant nontyphoidal Salmonellae, 2004-2009.

To evaluate trends in and risk factors for acquisition of antimicrobial-drug resistant nontyphoidal Salmonella infections, we searched Oregon surveillance data for 2004-2009 for all culture-confirmed cases of salmonellosis. We defined clinically important resistance (CIR) as decreased susceptibility to ampicillin, ceftriaxone, ciprofloxacin, gentamicin, or trimethoprim/sulfamethoxazole. Of 2,153 cases, 2,127 (99%) nontyphoidal Salmonella isolates were obtained from a specific source (e.g., feces, urine, blood, or other normally sterile tissue) and had been tested for drug susceptibility. Among these, 347 (16%) isolates had CIR. The odds of acquiring CIR infection significantly increased each year. Hospitalization was more likely for patients with than without CIR infections. Among patients with isolates that had been tested, we analyzed data from 1,813 (84%) who were interviewed. Travel to eastern or Southeast Asia was associated with increased CIR. Isolates associated with outbreaks were less likely to have CIR. Future surveillance activities should evaluate resistance with respect to international travel.

Salmonella gastroenteritis in children (clinical characteristics and antibiotic susceptibility): comparison of the years 1995-2001 and 2002-2008.

We document herein the prevalence and serotype distribution among Salmonella enterica strains isolated from children treated for diarrhea over two seven-year periods spanning 14 years. Four hundred and eight (1.38%) S. enterica cases were isolated among 29,601 diarrheal admissions. Among the Salmonella isolates, 63.7% were serogroup D and 29.9% were serogroup B. Overall, 21.7% of cases were under one year of age, with 2.1% being younger than three months. Bloody diarrhea was found in 18.8% of the cases. The resistance rates were 25.8%, 18.2%, 7.0%, 4.7%, and 0.3%, to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, ceftriaxone, and ciprofloxacin, respectively. In conclusion, our study has revealed that the predominance of Salmonella serogroup D continues. The clinical features of our patients were mostly mild, with no deaths or severe complications. While resistance to antimicrobial agents changes constantly, it is important to keep these strains under surveillance in order to formulate policies for the rational use of antimicrobial agents.

Salmonellosis outbreak due to Salmonella enteritidis phage type 14b resistant to nalidixic acid, Austria, September 2010.

We report on a salmonellosis-outbreak due to Salmonella Enteritidis phage type 14b resistant to nalidixic acid (S. Enteritidis PT14b Nx) among residents and employees of a student residence in Austria, September 2010. The outbreak was described and analysed by a retrospective cohort study, and microbiological environmental investigations were conducted to identify the outbreak source(s) and the reservoir of the outbreak strain. A total of 66 persons fulfilled the outbreak case definition including 14 laboratory-confirmed cases. Food specific cohort-analyses by day revealed that consumption of potato salad (RR: 1.65, 95%CI: 1.35­2.01, p=0.001) and a cheese-sausage cold plate (RR: 2.24, 95%CI: 1.29­3.88, p=0.002) on 14 September was associated with being an outbreak case. We hypothesised that cross-contamination with S. Enteritidis PT14b Nx positive eggs had occurred during preparation of the potato salad and cold plate as a result of preparing in parallel egg-containing breaded cutlets on 14 September. A traced laying hen holding in eastern Austria was identified as the sole source of the consumable eggs in the student residence. By applying the legally mandated sampling method for epidemiological-related laying hen farms (one pooled dust sample à 150g, two paired boot swabs cultured separately), the outbreak strain could not be detected. Our findings, that legally required sampling methods for laying hen farms failed to detect the causative pathogen in a laying hen holding, despite an epidemiological link, underline the request stated by the European Food Safety Authority Panel on Biological Hazards for a more sensitive sampling plan in epidemiologically-associated laying hen flocks.

Suitability of repetitive-DNA-sequence-based PCR fingerprinting for characterizing epidemic isolates of Salmonella enterica serovar Saintpaul.

Three molecular typing methods, repetitive-sequence-based PCR (rep-PCR) fingerprinting, plasmid profiling, and arbitrarily primed PCR fingerprinting, were used to characterize isolates of Salmonella enterica serovar Saintpaul. Most of the isolates were obtained from epidemic human cases of food-borne salmonellosis, together with some from the food material suspected to be the source of infection, and a few were obtained from other cases apparently not related to the epidemic. All three methods adequately discriminated the epidemic strain from other strains of the serovar. In addition several isolates from human cases which are not identical to the epidemic strain were found. These isolates therefore must have been responsible for some sporadic infections, which were only temporally related to the epidemic. These strains showed a high degree of similarity to a strain isolated from a turkey. rep-PCR fingerprinting with REP-Dt primers and primer ERIC1R, applicable even to crude cell lysates, offers an attractive choice as a primary method for the discrimination of various Salmonella serotypes as well as isolates within serotype Saintpaul.

Nationwide outbreak of human salmonellosis in Germany due to contaminated paprika and paprika-powdered potato chips.

Between April and September 1993, a nationwide outbreak of salmonellosis occurred in Germany which was traced to contaminated paprika and paprika-powdered potato chips. Of the estimated 1000 cases, children below 14 years were principally affected. Levels of 0.04-0.45 organisms per gram were found in the snacks. The infective dose was estimated at 4-45 organisms with an attack rate of 1 in 10,000 exposed persons. The unique feature of the outbreak was the variety of serovars involved. S. saintpaul, S. rubislaw and S. javiana were isolated during the same time period from paprika powder, spice mixtures, snacks and patients. Their clonal identity was confirmed by molecular typing methods. Furthermore, monophasic and non-motile strains of rare salmonella O-groups were isolated from both paprika products and patients. This is the largest documented outbreak due to contaminated spices which proved that even extremely low numbers of salmonellae adapted to the dry state were able to cause illness.

Large scale use of ciprofloxacin in the control of a Salmonella outbreak in a hospital for the mentally handicapped.

An outbreak of salmonella food poisoning occurred in a hospital for the mentally handicapped in July 1990. Salmonella enteritidis phage type 4 was identified in 101 patients and eight staff. Standard infection control measures were instituted. Ciprofloxacin was given to all resident patients and to all affected staff. The outbreak was rapidly controlled. There were no new cases after ciprofloxacin was started and there were no clinical relapses. Microbiological relapsers were retreated with ciprofloxacin. A gradual return to normal activity was possible and within two months the hospital was functioning normally. No salmonellae have been identified in the hospital since that time, confirming that the organism was eradicated, rather than just temporarily suppressed.

A national outbreak of salmonellosis from yeast flavoured products.

A national outbreak of salmonellosis caused by a rare serotype occurred between July and November 1989. A total of 40 cases of Salmonella manchester infection were identified by the PHLS Division of Enteric Pathogens with a further 7 cases reported from Scotland. The median age of those affected was one year. All strains from the outbreak carried a 70mDal plasmid with a distinctive restriction endonuclease. A statistical association was found between infection and consumption of nationally distributed savoury corn snacks. Samples of autolysed yeast powder and flavourings used in the manufacture of many processed foods were also found to be positive for S. manchester.