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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Mucosal competitive exclusion to reduce Salmonella in swine

A mucosal competitive exclusion culture has been shown to reduce or eliminate Salmonella spp. in poultry. Using similar techniques, a mucosal competitive exclusion culture from swine (MCES) was produced from the cecum of a 6-week-old pig. Suckling pigs were inoculated with 5 ml of MCES by oral gavage within 6 h postfarrowing (PF) and again at 24 h PE All pigs were challenged with 10(3) CFU of Salmonella Choleraesuis at 48 h PF by intranasal instillation, including pigs from two sows that had not been given MCES. Clinical signs and rectal swabs were monitored daily, and pigs were allowed to suckle throughout the experiment. All pigs underwent necropsy on day 7 PF, and presence of Salmonella was determined in both qualitative (10 tissues) and quantitative (two tissues) samples. Clinical signs were inapparent in all pigs throughout the experiment. Recovery of Salmonella from rectal swabs was variable. However, 28% of the gut tissues were positive from the MCES-treated pigs versus 79% from the control pigs. A 2- to 5-log10 reduction of Salmonella in the cecal contents or ileocolic junction was observed in the MCES-treated pigs when compared with the controls. These data indicate that use of MCES may be a useful approach for control of Salmonella.     

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.     

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.     

Reduction of Salmonella Typhimurium and Listeria monocytogenes on produce by trisodium phosphate

The application of trisodium phosphate (TSP) on produce against food-borne bacteria has not been extensively evaluated. This research studied the effect of 20 and 50 mg/ml TSP in reducing Salmonella Typhimurium (ST) and Listeria monocytogenes (Lm) on model produce (lettuce and peppers). Washed and air-dried Iceberg lettuce (3 × 3 cm²) and Jalapeno peppers (25–30 g) were spiked with S. Typhimurium or L. monocytogenes (～7 log₁₀ CFU/ml). Samples were treated with 20 or 50 mg/ml TSP, 200 mg/L sodium hypochlorite (for comparison with traditional washes) or water (control rinse) for 15 or 30 s. Treatments were immediately neutralized with trypticase soy broth (TSB) containing 30 mg/ml beef extract, serially diluted, plated on Xylose Lysine Tergitol 4 (XLT4) agar for ST and Tryptic Soy Agar (TSA) for Lm and incubated at 37 °C for 24–48 h. Results showed that 20 mg/ml TSP and 200 mg/L sodium hypochlorite caused 5–6 log₁₀ CFU/ml reduction in ST on both lettuce and peppers after 15 s and 30 s, while 50 mg/ml TSP reduced ST to undetectable levels on both produce at both contact times. For overnight Lm cultures, a mere reduction of 0.21 and 0.28 log₁₀ CFU/ml was obtained using 20 and 50 mg/ml TSP after even 5 min, while 200 mg/L sodium hypochlorite decreased Lm by ～1 log₁₀ CFU/ml after 30 s and 1 min on lettuce, and decreased pure culture Lm to undetectable levels after 3 min. Thus, 50 mg/ml TSP appears effective against ST with negligible effects against Lm, whose mode of action needs to be understood.     

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.     

Antimicrobial resistance of Salmonella enterica serovar Typhimurium isolated from cattle in Japan

The antimicrobial susceptibility of 144 Salmonella enterica serovar Typhimurium isolates collected from all over Japan between 1973 and 1998 were investigated. All the isolates exhibited resistance to four or more antimicrobials and 22 resistance patterns were observed. Isolates showing resistance patterns to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfonamides (Su) and tetracycline (T), which are typical resistance patterns for S. Typhimurium DT104 (DT104), were predominant. Thirty-six of the 68 isolates that exhibited resistance to five or more antimicrobials (ACSSuT+) were identified as DT104 by phage typing. Another 103 S. Typhimurium strains gathered from cattle between 1977 and 1999 in a limited area of Japan were analyzed for molecular epidemiological studies. Results using fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis suggest that clonal exchange of S. Typhimurium among cattle in Japan has occurred since 1992, and that contemporary strains show a remarkable degree of homogeneity with DT104 at a molecular level. The clonal replacement by DT104 affected the antimicrobial resistance pattern of S. Typhimurium from cattle in Japan.     

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%.