Susceptibility of food-borne bacteria to binary combinations of antimicrobials at selected a(w) and pH

AIM: To evaluate the antibacterial susceptibilities of food-borne bacteria to individual and binary mixtures of a synthetic antimicrobial agent with a natural phenolic compound. METHODS AND RESULTS: Antibacterial susceptibilities of Escherichia coli, Listeria innocua, Salmonella Typhimurium and Staphylococcus aureus to individual and binary mixtures of potassium sorbate with a phenolic compound (thymol, carvacrol, or eugenol) were evaluated, at selected water activity (a(w); 0.99 or 0.97) and pH (5.5 or 4.5). The bacteria studied were susceptible to the action of the antimicrobials individually with minimal inhibitory concentrations that varied from 800-ppm potassium sorbate for Staph. aureus at a(w) 0.99, and pH 5.5 to 100-ppm thymol or carvacrol for the four studied bacteria at a(w) 0.97 and pH 4.5. Several binary mixtures of potassium sorbate with thymol, carvacrol or eugenol inhibited bacterial growth. Antimicrobial agent inhibitory concentrations in the mixture varied among bacteria, additionally depending on the a(w) and the pH tested. CONCLUSIONS: Synergistic binary mixtures with fractional inhibitory concentration index <0.6 include 100- or 200-ppm potassium sorbate with 50- or 100-ppm thymol, carvacrol or eugenol. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic combinations could be useful in reducing the amounts of antimicrobials needed to inhibit growth, thus diminishing consumer concerns regarding chemical preservatives.     

The effect of bovine diet on Salmonella survival in synthetic abomasal fluid

Aims: To investigate the effect of diet on the survival of Salmonella in the bovine abomasum. Methods and Results: Five fistulated cows were randomly assigned to one of five diets denoted as: (i) 100% grass, (ii) grass + 5·3 kg DM concentrate, (iii) 100% grass silage, (iv) 100% hay and (v) maize/grass silage plus concentrates. Rumen fluid was harvested from each dietary treatment and inoculated with nonacid (NA) and acid‐adapted (AA) 5‐strain Salmonella cocktails. After 24‐h incubation period, Salmonella were acid challenged to synthetic abomasum fluid (SAF, pH 2·5) for 5 h to determine their resistance to low pH. The study found that the volatile fatty acids composition and the pH profile of bovine rumen fluid were significantly altered (P < 0·05) by some of the dietary treatments but not others. Regression analysis found that significantly higher numbers of acid‐adapted Salmonella survived in SAF after incubation in rumen fluid from diets 1, 2 and 4, but fewer significant differences were found between diets for nonacid–adapted Salmonella. The results suggest that the acid‐adapted cells were subjected to a higher level of cell injury than the nonadapted cells. Conclusions: Pre‐incubation in rumen fluid did influence the resistance of nonacid and acid‐adapted Salmonella to SAF but it was dependant on the dietary treatment fed to the cows. Significance and Impact of the Study: This study examined the use of diet, as a modulating factor to limit the bovine excretion of Salmonella with a view to providing a scientific basis for the design of dietary management controls in the future.     

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop‐mediated isothermal amplification

Aim: The objective of this study is to develop a serovar‐specific loop‐mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. Methods: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real‐time polymerase chain reaction (FQ‐PCR). Results: The results were as follows. (1) Serovar‐specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20 min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies μl^?1; thus, the sensitivity and specificity of this assay is similar to those of the FQ‐PCR. Conclusions: LAMP is a high‐throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. Significance and impact of the study: This is the first study involving the use of LAMP to detect Salmonella serovar‐specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.     

Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium

Quinolone resistance in Salmonella spp. is usually attributed to both active efflux and mutations leading to modification of the target enzymes DNA gyrase and topoisomerase IV. Here, we investigated the presence of mutations in the efflux regulatory genes of fluoroquinolone- and multidrug-resistant mutants of Salmonella enterica serovar Typhimurium (S. Typhimurium) selected in vitro with enrofloxacin that both carried a mutation in the target gene gyrA and overproduced the AcrAB efflux pump. No mutations were detected in the global regulatory loci marRAB and soxRS for the four strains studied. A mutation in acrR, the local repressor of acrAB, was found for two ciprofloxacin-resistant selected-mutants, leading to duplication of amino acids Ile75 and Glu76. Complementation experiments with wild-type acrR showed that the mutation identified in acrR partially contributed to the increase in resistance levels to several unrelated antibiotics. The acrR mutation also contributed to acrAB overexpression as shown by RT-PCR. Thus, this study underlines the role of an acrR mutation, in addition to the mutation in gyrA, in the fluoroquinolone and multidrug resistance phenotype of S. Typhimurium mutants, through overexpression of acrAB.     

Oxidation-reduction potential regulates RpoS levels in Salmonella Typhimurium

AIMS: The aim of this work was to investigate the connection between oxidation-reduction (redox) potential and stationary phase induction of RpoS in Salmonella Typhimurium. METHODS AND RESULTS: A lux-based reporter was used to evaluate RpoS activity in S. Typhimurium pure cultures. During growth of S. Typhimurium, a drop in the redox potential of the growth medium occurred at the same time as RpoS induction and entry into stationary phase. An artificially induced decrease in redox potential earlier during growth reduced the time to RpoS induction and Salmonella entered the stationary phase prematurely. In contrast, under high redox conditions, Salmonella grew unaffected and entered the stationary growth phase as normal, although RpoS induction did not occur. As a consequence, stationary phase cells grown in the high redox environment were significantly more heat sensitive (P < 0.05) than those grown under normal conditions. CONCLUSIONS: This work suggests that redox potential can regulate RpoS levels in S. Typhimurium and can thus, control the expression of genes responsible for thermal resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to manipulate RpoS induction and control stationary phase gene expression can have important implications in food safety. Early RpoS induction under low redox potential conditions can lead to enhanced resistance in low cell concentrations to inimical processes such as heat stress. Inhibition of RpoS induction would abolish stationary phase protective properties making cells more sensitive to common food control measures.     

The relationship between concentration of a dual marker strain of Salmonella Typhimurium in bovine faeces and its probability of detection by immunomagnetic separation and culture

AIMS: To modify a strain of Salmonella serotype Typhimurium to express unique marker traits and then define how the concentration of the marker in bovine faeces affects the probability of its detection by culture preceded by immunomagnetic separation (IMS). METHODS AND RESULTS: DNA encoding for the production of green fluorescent protein (gfp) and resistance to kanamycin was inserted into the bacterial chromosome of Salm. Typhimurium. Transposon insertion was demonstrated by Southern blot hybridization. Varying amounts of one electroporant (gfpSal-1) were inoculated into suspensions of bovine faeces and attempts made to isolate gfpSal-1 using a protocol based on pre-enrichment incubation, IMS and enrichment in selective media. Isolates of gfpSal-1 were differentiated from wild strains of Salmonella using fluorescence under u.v. light and expression of kanamycin resistance. A logistic and Gompertz function each derived from the dose-response data partially explained the observations with the fit of the Gompertz function judged to be superior. The 10, 50 and 90% limits of detection from the Gompertz function were estimated to be 1.92, 2.03 and 2.27 CFU g(-1) respectively. CONCLUSIONS: Reliance on the traditional concept of 'limit of detection' could introduce unacceptable errors in the interpretation of test findings when the concentration of Salm. Typhimurium in bovine faeces (pooled or individual) is below ca 3 CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The dose-response curve can be used to aid the design of protocols for detecting Salmonella in individual and pooled faecal specimens. The experiments demonstrate that both reporter genes in tandem are useful for studying the performance of culture-based methods for detecting pathogens in faeces.     

应用MUCAP试剂快速检测沙门氏菌

报告了用4-甲基伞形酮辛酯(4-Methylumbelliferyl-caprylate, MUCAP)快速检测沙门氏菌的特异性、敏感性和实用性。经HE,DHL,SS和麦康凯琼脂平板分离的65株沙门氏菌标准菌株和48株从食品中分离的沙门氏菌,用MUCAP测试均呈阳性反应;394株非沙门氏菌中呈阳性反应的假单胞菌、气单胞菌、邻单胞菌可通过氧化酶试验与沙门氏菌区分开;与粘质沙雷氏菌的交叉反应改用加1％蔗糖的分离平板也可排除。此方法的敏感性和特异性均达到97％以上,而且操作简便、快速,数分钟内即可完成。     

FTIR spectro-imaging of collagens for characterization and grading of gliomas

Collagens are a family of at least 30 protein types organized as networks. They constitute the main support material of cells under the form of extracellular matrix as well as for membranes in vessels, organs, and tissue compartments. Collagen network abnormalities are at the origin of many diseases, including myopathies and fibroses. The characterization of collagens remains an analytical challenge due to the insolubility of these molecules and the difficulty encountered in isolating given types without altering their structure or in maintaining network organization, which is critical to diagnosing related pathologies. We have proposed using a vibrational spectroscopy based imaging technique, namely Fourier-transform infrared (FTIR) imaging, for a spatially-resolved analysis of secondary structure of different collagen types in complex samples, and more specifically for characterizing gliomas. With newly developed spectral data treatments and chemometrics using secondary structure parameters of collagen proteins, FTIR imaging is now able to distinguish between several types. On this basis, gliomas have been investigated as specific collagen-rich tissues developing in a non-collagenous environment, providing high specificity to this FTIR imaging utilization. Here, we review the recent advances in this imaging approach for understanding glioma development, with FTIR imaging now being proposed as a molecular histopathology tool for clinicians.     

A single dose of recombinant Salmonella typhimurium induces specific humoral immune responses against heterologous Eimeria tenella antigens in chicken

Salmonella typhimurium vaccine strains were used as antigen delivery system for oral immunisation of chickens against two antigens of the coccidian parasite Eimeria tenella. The cDNAs of the known E. tenella proteins, SO7 and TA4, were isolated from total RNA and subcloned into the expression vectors pQE30 and pTECH2. Subcutaneous immunisation of chickens with Escherichia coli-expressed SO7 and TA4 revealed that both proteins were immunogenic. Both cDNAs were subcloned into plasmids of the pTECH2 vector system, which allows them to be expressed as fusion proteins with the highly immunogenic fragment C of the tetanus toxin under control of the anaerobically inducible nirB promoter. Plasmids were introduced into the S. typhimurium vaccine strains SL3261, C5aroD and C5htrA. SDS-PAGE and Western blot analysis revealed expression of both fusion proteins in all strains under anaerobic culture conditions. Three-week-old white leghorn chickens were orally immunised with 10(9) CFU per animal. The stability of the recombinant bacteria was revealed by recovery of viable Salmonella containing the respective plasmids from the liver of the immunised chickens at day 3 after inoculation. Specific serum IgG antibodies against the SO7-or TA4-antigens were detectable by ELISA 2 weeks after oral immunisation and remained for at least 6 weeks, while specific IgA antibodies were restricted to the bile of the birds. All chickens produced serum IgG and IgA to S. typhimurium lipopolysaccharides. Our data show that a single oral inoculation with recombinant S. typhimurium SL3261, C5aroD and C5htrA can induce specific antibody responses to heterologous Eimeria antigens in chickens, suggesting that recombinant Salmonella are a suitable delivery system for vaccines against Eimeria infections.