Inhibition of nalidixic acid-resistant salmonella on marinated chicken skin

Marination is widely used to enhance flavor and increase consumer acceptability of meat and poultry products. The impact of such marination on the safety and shelf life of poultry meat was evaluated in this study. A series of experiments were conducted to determine the efficacy of teriyaki and lemon pepper marinades against multiple strains of nalidixic acid (NAL)-resistant Salmonella. NAL-resistant Salmonella serovar (Typhimurium, Heidelberg, and Senftenberg) cultures were inoculated onto chicken skin at 0.6 to 3.14 log CFU/g in a 12-well titer plate. Inoculated chicken skin was exposed to teriyaki or lemon pepper marinades for up to 32 h and stored at 4 or 25°C to determine the prevalence of Salmonella. To determine Salmonella survival, a three-strain cocktail of Salmonella was inoculated at low (ca. 4 log CFU/g) and high (8 log CFU/g) levels onto chicken skin that was then marinated with either teriyaki or lemon pepper marinade for up to 32 h and stored at 4 or 25°C. Prevalence of Salmonella was significantly reduced (P ≤ 0.05) by teriyaki marinade at all levels of contamination regardless of storage temperature. Lemon pepper marinade reduced Salmonella prevalence (P ≤ 0.05) at low levels of contamination (101 and 102 CFU/g), whereas no significant effect (P > 0.05) was observed at higher levels of contamination. Marination of chicken skin with teriyaki marinade greatly reduced Salmonella prevalence and survival (P ≤ 0.05) regardless of the storage temperature, indicating the antimicrobial potential of this marinade for poultry and meat products.     

Characterization of the quinolone resistance mechanism in foodborne Salmonella isolates with high nalidixic acid resistance

Sixteen Salmonella strains resistant to nalidixic acid isolated from kimbab, the most popular ready-to-eat (RTE) food in Korea, and chicken meat were selected for this study. The resistant strains were shown to have high minimal inhibitory concentrations (MICs) against nalidixic acid (512 ~ 4096 μg/mL). Among them, 4 Salmonella enterica serovar Haardt isolates showed multi-drug resistance (MDR) patterns with reduced susceptibility to fluoroquinolone (0.5 μg/mL of ciprofloxacin MICs). The mechanisms of quinolone resistance in the nalidixic acid resistant strains were characterized by PCR and sequence analysis. The presence of plasmid-mediated quinolone resistance (PMQR) genes and amino acid changes in the quinolone resistance determining region (QRDR) were investigated by PCR-based detection and sequencing, and the efflux pump inhibition test was also done using phe-arg-β-naphthylamide (PAβN). Although PMQR genes were not detected in any of the tested strains, the QRDR mutations were found in this study: single mutation in gyrA (Asp87Tyr, Asp87Gly, and Asp87Asn), double mutations in gyrA (Ser83Thr) and parC (Thr57Ser), and single mutation in parC (Thr57Ser). MICs of nalidixic acid were reduced by 2- to 32-folds by the efflux pump inhibitor, PAβN. Pulsed-field gel electrophoresis (PFGE) was carried out to confirm the epidemiological relationship between the nalidixic acid resistant strains. The PFGE patterns were classified into 6 groups at cutoff level of 70 ~ 100% correlation on the dendrogram. Some strains of serotype Haardt and Enteritidis showed several values of genomic identity in accordance with strains, sources, and isolation year. We suggest that point mutation on QRDR and efflux pump systems involved in antimicrobials had independent effects on drug-resistance regardless of bacterial genomic variation.     

Development and Characterization of Monoclonal and Polyclonal Antibodies Against Salmonella enterica Typhimurium for Biosensor Detection

ABSTRACT: For the construction of a biosensor to specifically and effectively detect Salmonella enterica Typhimurium, a monoclonal antibody (mAb) 1B4 and a polyclonal antibody (pAb) S48 were developed against purified outer membrane proteins (OMPs) of Salmonella enterica Typhimurium. Gel electrophoresis data indicated that the apparent molecular weight of the main OMP was 55 kD. The mAb 1B4 strongly reacted to Salmonella enterica Typhimurium and Salmonella enterica Paratyphi and had no cross-reaction to other gram-negative and gram-positive bacteria that were tested. The pAb S48 also showed high reactivity to Salmonella enterica Typhimurium, but had cross-reactivity with other tested bacteria. Because of the high specificity and sensitivity of 1B4 against S. enterica Typhimurium, it was immobilized onto a sensor platform to capture bacteria. The captured bacteria were visualized byalight microscope fortheir detection. The detection limit of the biosensorwas 1 × 10² colony forming units/mL in bacterial culture and in a milk sample, and no other bacteria interfered with the binding of targeted cells. Therefore, a biosensor was developed that would rapidly detect Salmonella in the laboratory and in a food processing plant.     

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.     

四川省鸭和猪源鼠伤寒沙门菌脉冲场凝胶电泳分型与耐药比较分析

目的比较研究四川省鸭和猪源鼠伤寒沙门菌脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分型与耐药特征,为分析鼠伤寒沙门菌食品来源提供支持。方法将鸭和猪源监测中分离的47株鼠伤寒沙门菌进行PFGE分型,并采用最小抑菌浓度法测定9种药物敏感性。结果鸭和猪源中分离的鼠伤寒沙门菌在PFGE分型和耐药上存在差异。鸭源菌株聚集于簇I,而猪源菌株聚集于簇II。簇II菌株对氨苄西林、环丙沙星、氯霉素、庆大霉素、四环素、复方新诺明6种药物的耐药率高于簇I菌株。不同的PFGE分型鼠伤寒沙门菌在监测地区和耐药谱存在差异。SCSTm-10型菌株均分离于绵阳地区,SCSTm-11型主要分离于资阳地区。SCSTm-10型菌株对复方新诺明和环丙沙星的耐药率高于SCSTm-11型菌株。结论 PFGE分型可以获取四川省鼠伤寒沙门菌分离来源、分离地点和耐药的聚类信息,为鼠伤寒沙门菌食物中毒提供流行病学调查支持。     

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。     

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.     

Characterization of antimicrobial resistance in Salmonella enterica serovar Typhimurium isolates from food animals in the U.S.

Salmonella enterica subspecies enteric serovar Typhimurium is the second most common serovar implicated in human diseases in the United States. In this study, 120 S. Typhimurium isolates from animal sources were characterized by antimicrobial susceptibility testing, antimicrobial resistance gene detection and plasmid analysis. Overall, 94 (78%) of the isolates were resistant to one or more antimicrobials and 63 (53%) were resistant to at least five antimicrobials. Resistance was most commonly observed to streptomycin (62%), sulfisoxazole (62%), or tetracycline (61%). When resistance was detected, a corresponding resistance gene was detected in 89% of cases. Class 1 integrons were detected in 51 isolates, all which contained the aadA2 gene. A plasmid Inc group was detected in 68 (57%) isolates. Thirty nine (57%) of these isolates were resistant to 5 or more antimicrobials.     

Distribution of Salmonella in swine production ecosystems

The objective of this 2-year field survey was to sample multiple ecological compartments within swine production systems to identify potential sources of Salmonella infection for swine. Twelve single-site production systems within Illinois were identified by slaughter sampling to have detectable Salmonella in swine and therefore selected for study. There were four visits to each farm during a 5-month period. Fecal samples were obtained from swine and other wild and domestic mammals. Arthropods and environmental samples of feed, water, pen floors, boots, and bird feces were also collected. All 8,066 samples obtained were cultured to detect Salmonella. Salmonella was detected on 11 of the 12 farms. There were 206 positive cultures, including samples from swine (83), pen floors (54), boots (32), flies (16), mice (9), cats (3), and birds (3). Swine shedding Salmonella in feces were detected on 9 of the 12 farms. The more Salmonella-abundant ecological compartments were cats (12% of samples positive), boots (11%), bird feces (8%), flies (6%), and mice (5%); 2.1% of 4,024 swine samples were positive. All 221 feed samples were negative for Salmonella. There was a correlation between a farm having a high prevalence of shedding Salmonella in pigs and a high abundance on pen floors, flies, and boots. The most common serotypes detected were Derby, Agona, Worthington, and Uganda, which were distributed throughout the ecosystem, suggesting widespread transmission across ecological compartments. The ubiquitous distribution of Salmonella suggests that an effective control strategy must target multiple compartments of the swine production ecosystem.     

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.     

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.     

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.     

Effects of nitrate or nitro supplementation, with or without added chlorate, on Salmonella enterica serovar Typhimurium and Escherichia coli in swine feces

The effects of coincubating the active agent of an experimental chlorate product with nitrate or select nitro compounds, possible inducers and competing substrates for the targeted respiratory nitrate reductase, on concentrations of experimentally inoculated Salmonella enterica serovar Typhimurium and indigenous Escherichia coli were determined. Studies were completed in swine fecal suspensions as a prelude to the administration of these inhibitors to pigs. Results confirmed the bactericidal effect of chlorate (5 to 10 mM) against these fecal enterobacteria, reducing (P < 0.05) concentrations by > 2 log CFU ml(-1) after 3 to 6 h of incubation. An effect (P < 0.05) of pH was observed, with considerable regrowth of Salmonella and E. coli occurring after 24 h of incubation in suspensions buffered to pH 7.1 but not in suspensions buffered to pH 6.5 or 5.6. A 24-h coincubation of fecal suspensions with 5 to 10 mM chlorate and as little as 2.5 mM nitrate or 10 to 20 mM 2-nitro-1-propanol, 2-nitroethanol, and, sometimes, nitroethane decreased (P < 0.05) Salmonella but not necessarily E. coli concentrations. 2-Nitro-1-propanol and 2-nitroethanol exhibited inhibitory activity against Salmonella and E. coli by an undetermined mechanism, even in the absence of added chlorate.     

Survival of Campylobacter jejuni and Salmonella enterica Typhimurium in vacuum-packed, moisture-enhanced pork

The abilities of Campylobacter jejuni and Salmonella enterica Typhimurium to survive in vacuum-packaged, moisture-enhanced pork stored at 4 or 10°C were examined. Pork loins were surface inoculated with either C. jejuni or Salmonella Typhimurium and then moisture enhanced to a target of 10 or 20%. The enhanced pork loins were sliced 1 cm thick and vacuum packaged. A pork loin without moisture enhancement was sliced and vacuum packaged as a control. Samples were collected, plated, and the numbers of surviving organisms were determined periodically during storage at 4 and 10°C. The numbers of C. jejuni or Salmonella Typhimurium in samples with different moisture enhancement levels were similar (P > 0.05). No significant differences (P > 0.05) in C. jejuni counts were observed between samples at 10°C and those at 4°C. In contrast, the numbers of Salmonella Typhimurium in samples at 10°C had significantly (P < 0.05) increased (0.41 log CFU/g) from those at the refrigerated temperature of 4°C. Vacuum storage at 4 and 10°C for 28 days did not result in dramatic reductions in the mean numbers of C. jejuni and Salmonella Typhimurium. Our findings indicate that vacuum packaging under chilled conditions will not add substantially to safety for moisture-enhanced pork. Strict hygienic practices or the implementation of decontamination technologies is recommended.     

Survival and infectivity of Salmonella choleraesuis in swine feces.

Many serotypes of Salmonella survive well in the environment. Conversely, it is believed that Salmonella Choleraesuis, the host-adapted serotype of swine, does not survive well outside the host. We examined the survival capability of Salmonella Choleraesuis in swine feces. Six pigs were infected with Salmonella Choleraesuis and feces were collected and pooled on days 2, 4, 7, and 10 postinoculation (PI). Feces were stored in a wet and a dry form, and survival was measured over 13 months. Salmonella Choleraesuis was recovered from wet feces through 3 months of storage. In a desiccated (dry) form, Salmonella Choleraesuis was recovered from at least 13 months. Salmonella Choleraesuis shed from swine prior to 4 days PI did not survive as well as that shed 4 days PI or later. We also examined the infectivity of Salmonella Choleraesuis resident in dry feces. Six- or 13-week-old pigs were inoculated with dry feces that had been stored either 2 months or 4 months, respectively. Pigs were inoculated either intranasally or by mixing dry feces with the swine ration. Although clinical signs were mild, Salmonella Choleraesuis was widely disseminated among the tissues of all the pigs inoculated. This study demonstrates that Salmonella Choleraesuis remains viable and infective in the environment. Therefore, contaminated fecal matter can serve as a reservoir for Salmonella Choleraesuis as well as other Salmonella spp. Control measures must consider this environmental reservoir as a source of new infections.     

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.