Bacteriophage control of Salmonella Typhimurium in milk

Food Science and Biotechnology - This study was designed to evaluate the effect of bacteriophage P22 on the inhibition of growth of Salmonella Typhimurium. The P22 belongs to Podoviridae family...     

Multidrug-resistant Salmonella Typhimurium DT104 in poultry

Salmonella Typhimurium isolates from feed ingredients or poultry sources isolated during 1995 to 1997 from different geographical locations within Minnesota were examined for the presence of Salmonella Typhimurium definitive type 104 (DT104). Antibiotic susceptibility studies indicated that 15 of 50 isolates of Salmonella Typhimurium had an antibiotic resistance pattern (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) that is usually observed with multidrug-resistant Salmonella Typhimurium DT104. Of the 15 isolates showing the antibiotic resistance pattern, 8 isolates were phage type 104, 3 isolates were typed as phage type 104 complex, and the remaining 4 isolates belonged to phage types 193, 81, and 126. DT104 was recovered from both feed ingredients and poultry samples. Of the seven feed ingredient-associated Salmonella Typhimurium isolates, four were DT104, whereas only 7 of 43 poultry-associated Salmonella Typhimurium isolates were DT104. A repetitive sequence-based polymerase chain reaction (rep-PCR) of 50 isolates of Salmonella Typhimurium representing 13 phage types identified seven distinct fingerprint profiles. No correlation between phage type and rep-PCR type was noticed. Eleven Salmonella Typhimurium isolates belonging to DT104 and its complex were grouped into two closely related rep-PCR types.     

鼠伤寒沙门菌对抗生素和消毒剂耐药性研究

目的检测302株食品源、动物源和人源鼠伤寒沙门菌(Salmonella enterica Typhimurium)对抗生素和消毒剂的耐药表型,测定消毒剂耐药基因型,并分析鼠伤寒沙门菌对抗生素和消毒剂耐药的相关性。方法纸片扩散法测定鼠伤寒沙门菌对16种抗生素的耐药性,微量肉汤稀释法测定4种消毒剂的最小抑菌浓度(minimal inhibitory concentrations, MICs),聚合酶链式反应(polymerase chain reaction, PCR)检测消毒剂的耐药基因。结果鼠伤寒沙门菌分离株中96.03%(290/302)至少对一种抗生素有耐药性,79.80%(241/302)是多重耐药(multidrug resistant, MDR)菌株。分离株对链霉素的耐药率最高(78.81%,238/302),其次是磺胺复合物(78.15%,236/302)、四环素(75.50%,228/302)、氨苄青霉素(71.85%,217/302),所有分离株均对头孢吡肟和亚胺培南敏感。食品源鼠伤寒沙门菌对磺胺复合物、四环素、氨苄西林、萘啶酸和庆大霉素的耐药率均高于动物源和人源鼠伤寒沙门菌,差异有统计学意义(P0.05);动物源鼠伤寒沙门菌对环丙沙星和氧氟沙星的耐药率高于食品源和人源鼠伤寒沙门菌,差异有统计学意义(P0.05);人源鼠伤寒沙门菌对头孢噻肟和头孢他啶的耐药率高于食品源和动物源分离株。消毒剂苯扎氯铵、三氯生、三氯异氰尿酸和聚维酮碘对鼠伤寒沙门菌分离株的MICs范围分别为2～64、0.031 25～1、32～1 024和256～1 024 mg/L。食品源与动物源鼠伤寒沙门菌对苯扎氯铵、三氯生和聚维酮碘的耐药率高于人源鼠伤寒沙门菌,差异有统计学意义(P0.05)。分离株中消毒剂耐药基因qacEΔ1、sugE(p)和qacE检出率分别为56.95%(172/302)、20.53%(62/302)和2.65%(8/302),qacEΔ1基因与鼠伤寒沙门菌对β-内酰胺类、氨基糖苷类、四环素类、氟喹诺酮类、磺胺类、氯霉素类和喹诺酮类的耐药性显著相关(P0.01)。结论鼠伤寒沙门菌对抗生素耐药普遍,多重耐药率较高,并表现对消毒剂的耐药,鼠伤寒沙门菌对抗生素和消毒剂耐药存在相关性。     

热辅助超声波处理中VBNC态鼠伤寒沙门氏菌产生及其机制

为了研究热辅助超声波(TS)处理中鼠伤寒沙门氏菌进入活的非可培养态(VBNC)的情况及可能机制,本文采用RT-qPCR与平板计数法检测其VBNC态发生情况,采用ESR方法定性、定量检测TS处理中产生自由基并用数学模型分析其与VBNC态发生率指数之间的关系。结果表明:TS处理强度与VBNC态鼠伤寒沙门氏菌产生及自由基强度有重要影响。经TS处理,在BPY培养基中主要产生3种自由基,包括以碳为中心自由基、羟基自由基与氢质子自由基。自由基强度与VBNC态发生率指数之间高度相关,可用Boltzmann模型进行拟合和验证。以丙酮酸钠作为自由基清除剂,验证了自由基对鼠伤寒沙门氏菌进入VBNC态具有重要作用。本研究结果对于TS处理技术的应用具有指导意义。     

Biofilm formation and the survival of Salmonella Typhimurium on parsley

Although several studies provide evidence that the formation of biofilms by human pathogens on plant tissue is possible, to date there is no direct evidence that biofilms enhance the resistance of plant-associated pathogens to disinfectants or biocides. We hypothesized that biofilm formation would enhance the adhesion and survival of Salmonella on leafy vegetables. To test our hypothesis, we compared the adhesion and persistence of Salmonella Typhimurium and its biofilm-deficient isogenic mutant. Following inoculation of parsley and rinsing with water or chlorine solution, both strains had similar survival properties, and up to 3-log reduction were observed, depending on chlorine concentration. This indicates that the biofilm matrix of Salmonella likely does not play a significant role in initial adhesion and survival after disinfection. After a week of storage the biofilm producing strain survived chlorination significantly better than the biofilm-deficient mutant. However, the recovery of the mutant was still elevated, indicating that although the biofilm matrix has a role in persistence of Salmonella after chlorination treatment of parsley, this is not the most important mechanism, and other mechanisms, probably the ability to penetrate the plant tissue or the pre-existing biofilms, or production of different polysaccharides other than cellulose, provide the protection.     

Effectiveness of lemon juice in the elimination of Salmonella Typhimurium in stuffed mussels

Street foods are becoming more and more prominent in countries all over the world. There are many reports of disease due to consumption of street foods contaminated by pathogens. With the modern trend toward more natural preservatives, the use of organic acids can achieve a good microbiological safety in food. In the present study, stuffed mussels were inoculated with Salmonella Typhimurium suspension to provide initial populations of approximately 6 and 3 log CFU/g. After inoculation, samples were treated with fresh lemon juice and lemon dressing for 0, 5, and 15 min, and pathogens were enumerated by using direct plating on brilliant green agar. Treatment of stuffed mussels inoculated at high inoculum level, with lemon juice and lemon dressing for different exposure times caused reduction ranging between 0.25 and 0.56 log CFU/g and 0.5 and 0.69 log CFU/g, respectively, whereas in stuffed-mussel samples inoculated at low level, lemon juice and lemon dressing caused 0.08 to 0.25 log CFU/g and 0.22 to 0.78 log CFU/g reductions, respectively. Results of the study showed that both lemon juice and lemon dressing used as flavoring and acidifying agents for stuffed mussels caused slight decrease in Salmonella Typhimurium as an immediate inhibitor, but this effect increased by time. However, treatment of stuffed mussels with the inhibitors until 15 min is not enough to prevent Salmonella Typhimurium outbreaks related to stuffed mussels.     

Inactivation of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg by ultrasonic waves under pressure

The resistance of Salmonella Enteritidis (ATCC 13076), Salmonella Typhimurium (ATCC 13311), and Salmonella Senftenberg 775W (ATCC 43845) to ultrasonic waves under pressure treatments, at sublethal (manosonication) and lethal temperatures (manothermosonication) in citrate-phosphate buffer and in liquid whole egg was investigated. The influence of treatment parameters on the inactivation rate of manosonication was also studied. Decimal reduction times (Dt) of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg 775W corresponding to a heat treatment at 60 degrees C in pH 7 buffer and in liquid whole egg were 0.068, 0.12, and 1.0 min for buffer, and 0.12, 0.20, and 5.5 min for liquid whole egg. Those corresponding to a manosonication treatment (117 microns, 200 kPa, 40 degrees C) in both media were 0.73, 0.78, and 0.84 min, and 0.76, 0.84, and 1.4 min, respectively. When the amplitude of ultrasonic waves was increased linearly, the inactivation rate of manosonication increased exponentially. The inactivation rate also increased when pressure was raised. However, the magnitude of this increase was progressively smaller at higher pressures. The magnitude of the influence of the amplitude of ultrasonic waves and static pressure on the inactivation rate of manosonication was the same in the three serotypes investigated. Whereas a heat treatment at 60 degrees C only attained a 1/2-log cycle reduction in the number of Salmonella Senftenberg 775W survivors, a manothermosonication treatment (117 microns and 200 kPa) at this temperature attained a 3-log cycle reduction.     

Piezoelectric Flow Injection Analysis Biosensor for the Detection of Salmonella Typhimurium

ABSTRACT: Piezoelectric biosensors have the potential to provide direct detection of food contaminants, such as pathogens. In this study, Protein A antibody immobilization was used for the activation of the piezoelectric biosensor to detect Salmonella typhimurium. The overall system consisted of a new design for a flow cell and flow injection analysis system. The flow cell made possible a baseline stability of ± 1 Hz out of 5 MHz for hours. The sensor had responses of 5 to 65 Hz in 30 min with R²= 0.95 for S. typhimurium concentrations of 10⁷to 10⁹ CFU/ml under continuous flow, and 3 to 75 Hz in 40 min with R²= 0.96 for S. typhimurium concentrations of 10⁶ to 10¹⁰ CFU/ ml under stop flow. Cross-reactivity tests were performed with nonpathogenic Escherichia coli, Escherichia coli O157:H7, Listeria monocytogenes , and Vibrio parahaemolyticus and showed less than 10% response.     

过氧化氢处理中鼠伤寒沙门氏菌VBNC态形成及其机制解析

为确定过氧化氢的有效杀菌浓度,以鼠伤寒沙门氏菌为目标微生物,借助分子生物学手段,探究过氧化氢对自来水中鼠伤寒沙门氏菌活性和可培养性的影响及其活的非可培养状态(viable but non-culturable,VBNC)的形成原因。结果表明:较低过氧化氢浓度(0～15 mmol/lL)处理可达到部分杀菌的效果,1 mol/L以上过氧化氢可完全灭菌。而15 mmol/L过氧化氢处理导致4.08 lg(CFU/mL)鼠伤寒沙门氏菌全部进入VBNC状态。该状态的沙门氏菌形态和结构改变,过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶几乎完全被钝化,处理中产生的自由基强度在0～30 min内与VBNC发生系数呈正相关。     

Application of a bacteriophage cocktail to reduce Salmonella Typhimurium U288 contamination on pig skin

Multidrug-resistant Salmonella Typhimurium U288 is a significant pathogen of pigs, accounting for over half of all outbreaks on UK pig production premises. The potential of this serovar, and other salmonellae, to enter the food chain during the slaughtering process requires that efforts be made to reduce the prevalence of these bacteria at both the pre- and post-harvest stages of production. A bacteriophage cocktail (PC1) capable of lysing various Salmonella enterica serovars was designed using the broad host-range phage Felix 01, and three phages isolated from sewage. PC1 applied to pig skin experimentally-contaminated with U288 achieved significant reductions (P < 0.05) in Salmonella counts when stored at 4 °C over 96 h. Reductions of > 1 log₁₀ unit were observed when the ratio of phage applied was in excess of the bacterial concentration. The treatment was found to be effective at a multiplicity of infection (MOI) of 10 or above, with no significant reductions taking place when the MOI was less than 10. Under these conditions U288 counts of log₁₀ 4.1-4.3 CFU were reduced to undetectable levels following the application of PC1 to pig skin (> 99% reduction). These data suggest phage cocktails could be employed post-slaughter as a means to reduce Salmonella contamination of pig carcasses.     

Characterization of nalidixic acid-resistant and fluoroquinolone-reduced susceptible Salmonella Typhimurium in swine

From 2001 to 2008, a total of 27 isolates of Salmonella enterica serovar Typhimurium were obtained from 930 swine. All 27 isolates were resistant to streptomycin and tetracycline. Seventeen isolates were multidrug resistant to more than three antimicrobial agents. Seven of these multidrug-resistant isolates were pentaresistant to ampicillin, chloramphenicol, streptomycin, tetracycline, and nalidixic acid. Among 27 isolates, 14 isolates (51.8 %) were nalidixic acid resistant (MIC, ≥128 μg/ml) and had reduced susceptibility to various quinolones (MIC, 0.125 to 2 μg/ml). When quinolone resistance-determining regions in the gyrA and gyrB genes of these isolates were sequenced, 13 isolates had Asp87→Tyr mutations and 1 isolate had Asp87→Gly mutation in the quinolone resistance-determining region of gyrA, whereas no mutation was found in gyrB. Genes for qnrA, qnrB, and qnrS were not detected by PCR with specific primers. Pulsed-field gel electrophoresis of genomic DNA digested with Xba I showed two patterns suggesting a clonal spread of Salmonella Typhimurium in swine in Korea.     

Effect of guava extracts on heat resistance of Salmonella Typhimurium

The effect of exposure of Salmonella Typhimurium to guava extracts on bacterial heat resistance was investigated. After exposure to guava extracts for 3 h, the heat resistance of S. Typhimurium was determined at 57°C. Exposure of cells to guava extracts decreased D-values in the range of 18% to 52%, compared with unexposed cells. The lowest D-value was observed in cell exposed to acetone extracts of guava fruits. Exposure to guava extracts can make cells more sensitive to heat treatment.     

Viability of Listeria monocytogenes and Salmonella Typhimurium after isochoric freezing

Isochoric freezing, different from isobaric (conventional) freezing, allows for storage below freezing temperatures without significant damage from ice formation. While several types of tissues have been successfully stored in sub-zero isochoric conditions, it is unknown how isochoric freezing affects pathogenic microorganisms. Thus, the objective of this study was to investigate the survival of Salmonella Typhimurium and Listeria monocytogenes at below freezing storage (<0°C) in isochoric conditions. Tested conditions included storage at −4, −7, and −15°C for 24 hr and at −15°C for 1, 2, 3, 6, 12, and 24 hr. A comparison of bacterial survival during isobaric freezing was included with every trial. Additionally, bacterial cells were examined for morphological damage using transmission electron and field-emission scanning electron microscopes. Isochoric freezing at −15°C for 24 hr reduced both species of bacteria down to unrecoverable levels and maximum efficacy achieved after the 6 hr timepoint for L. monocytogenes and the 12 hr timepoint for S. Typhimurium. When viewed using electron microscopy, S. Typhimurium cells were noticeably disfigured with regions of cytosol separated from the cell wall. The results of this study demonstrate that isochoric freezing is capable of substantial levels of pathogen reduction. Unlike conventional nonthermal interventions, isochoric freezing does not require additional devices such as elevated pressure machines or pulsed electric fields and can be achieved with simple, inexpensive, rigid closed volume containers such as household freezers or commercial cold storage facilities.     

Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs

Despite washing and decontamination, outbreaks linked to consumption of fresh or minimally-processed leafy greens have been increasingly reported in recent years. In order to assure the safety of produce it is necessary to gain knowledge regarding the exact routes of contamination. Leaf internalization through stomata was previously reported as a potential route of contamination, which renders food-borne pathogens protected from washing and disinfection by sanitizers. In the present study we have examined the incidence (percentage of microscopic fields harboring ≥ 1 GFP-tagged bacteria) of Salmonella Typhimurium on the surface and underneath the epidermis in detached leaves of seven vegetables and fresh herbs. The incidence of internalized Salmonella varied considerably among the different plants. The highest incidence was observed in iceberg lettuce (81 ± 16%) and arugula leaves (88 ± 16%), while romaine (16 ± 16%) and red-lettuce (20 ± 15%), showed significantly lower incidence (P < 0.05). Internalization incidence in fresh basil was 46 ± 12%, while parsley and tomato leaves demonstrated only marginal internalization (1.9 ± 3.3% and 0.56 ± 1.36%, respectively). Internalization of Salmonella in iceberg lettuce largely varied (0-100%) through a 2 year survey, with a higher incidence occurring mainly in the summer. These results imply that Salmonella internalization occurs in several leafy vegetables and fresh herbs, other than iceberg lettuce, yet the level of internalization largely varies among plants and within the same crop. Since internalized bacteria may evade disinfection, it is of great interest to identify plants which are more susceptible to bacterial internalization, as well as plant and environmental factors that affect internalization.     

Dispersal of Salmonella Typhimurium by rain splash onto tomato plants

Outbreaks of Salmonella enterica have increasingly been associated with tomatoes and traced back to production areas, but the spread of Salmonella from a point source onto plants has not been described. Splash dispersal by rain could be one means of dissemination. Green fluorescent protein-labeled, kanamycin-resistant Salmonella enterica sv. Typhimurium dispensed on the surface of plastic mulch, organic mulch, or soil at 10? CFU/cm2 was used as the point source in the center of a rain simulator. Tomato plants in soil with and without plastic or organic mulch were placed around the point source, and rain intensities of 60 and 110 mm/h were applied for 5, 10, 20, and 30 min. Dispersal of Salmonella followed a negative exponential model with a half distance of 3 cm at 110 mm/h. Dispersed Salmonella survived for 3 days on tomato leaflets, with a total decline of 5 log and an initial decimal reduction time of 10 h. Recovery of dispersed Salmonella from plants at the maximum observed distance ranged from 3 CFU/g of leaflet after a rain episode of 110 mm/h for 10 min on soil to 117 CFU/g of leaflet on plastic mulch. Dispersal of Salmonella on plants with and without mulch was significantly enhanced by increasing rain duration from 0 to 10 min, but dispersal was reduced when rainfall duration increased from 10 to 30 min. Salmonella may be dispersed by rain to contaminate tomato plants in the field, especially during rain events of 10 min and when plastic mulch is used.     

Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars

Salmonella enterica serovar Typhimurium is a major foodborne pathogen throughout the world. Until now, the specific target genes for the detection and identification of serovar Typhimurium have not been developed. To determine the specific probes for serovar Typhimurium, the genes of serovar Typhimurium LT2 that were expected to be unique were selected with the BLAST (Basic Local Alignment Search Tool) program within GenBank. The selected genes were compared with 11 genomic sequences of various Salmonella serovars by BLAST. Of these selected genes, 10 were expected to be specific to serovar Typhimurium and were not related to virulence factor genes of Salmonella pathogenicity island or to genes of the O and H antigens of Salmonella. Primers for the 10 selected genes were constructed, and PCRs were evaluated with various genomic DNAs of Salmonella and non-Salmonella strains for the specific identification of Salmonella serovar Typhimurium. Among all the primer sets for the 10 genes, STM4497 showed the highest degree of specificity to serovar Typhimurium. In this study, a specific primer set for Salmonella serovar Typhimurium was developed on the basis of the comparison of genomic sequences between Salmonella serovars and was validated with PCR. This method of comparative genomics to select target genes or sequences can be applied to the specific detection of microorganisms.     

A risk characterization model of Salmonella Typhimurium in Irish fresh pork sausages

A second-order simulation model was built to estimate the risk of Salmonella Typhimurium associated with the consumption of Irish fresh pork sausages. To select appropriate hazard characterization models, an initial appraisal of the current dose-response models was conducted. The cooking modality of grilling was associated with a higher mean risk of infection per serving (1.399 × 10^? 6; 95% CI: 7.54 × 10^? 7–2.65 × 10^? 6) than frying (6.246 × 10^? 7, 95% CI: 2.78 × 10^? 7–1.17 × 10^? 6). When the risk was extrapolated over the consumption in a year period, the mean risk of infection increased considerably to 8.541 × 10^? 5 with an expected number of infections and illnesses of 184.3 (95% CI: 26–664) and 17 (95% CI: 2–63), respectively. Results highlighted the importance of consumer education, as scenario analysis predicted that, for the current level of Salmonella in pork sausage, decreasing the product's cold storage by approximately 8 h and cooking for an additional half minute can reduce the current risk level by ～ 50%.