Survival of Salmonella Under Heat Stress is Associated with the Presence/Absence of CRISPR Cas Genes and Iron Levels.

Clustered regularly interspaced short palindromic repeats (CRISPR) cas genes have been linked to stress response in Salmonella. Our aim was to identify the presence of CRISPR cas in Salmonella and its response to heat in the presence of iron. Whole genomes of Salmonella (n = 50) of seven serovars were compared to identify the presence of CRISPR cas genes, direct-repeats and spacers. All Salmonella genomes had all cas genes present except S. Newport 2393 which lacked these genes. Gene-specific primers were used to confirm the absence of these genes in S. Newport 2393. The presence/absence of CRISPR cas genes was further investigated among 469 S. Newport genomes from PATRIC with 283 genomes selected for pan-genome analysis. The response of eleven Salmonella strains of various serovars to gradual heat in ferrous and ferric forms of iron was investigated. A total of 32/283 S. Newport genomes that lacked all CRISPR cas genes clustered together. S. Newport 2393 was the most heat-sensitive strain at higher iron levels (200 and 220 pm) in ferrous and ferric forms of iron. The absence of CRISPR cas genes in S. Newport 2393 may contribute to its increase in heat sensitivity and iron may play a role in this. The high reduction in numbers of most Salmonella strains exposed to heat makes it unfeasible to extract RNA and conduct transcription studies. Further studies should be conducted to validate the survival of Salmonella when exposed to heat in the presence/absence of CRISPR cas genes and different iron levels.

Salmonella survival after exposure to heat in a model meat juice system.

The effect of heat against eleven Salmonella strains in model meat juices was examined. Juices from beef, lamb and goat were made from either the fatty layer (FL), muscle (M) or a mixture of both (FLM). The pH of each FLM sample was altered to match the pH of PBS and vice versa to determine the pH effect on the survival of Salmonella against the effect of heat. Salmonella were exposed to either gradual heating to 70 °C in FLM, M and FL or heat shock at 70 °C for 5 min in FLM. Fat, fatty acid profile and iron content of the juices were determined. Gradual heat treatment significantly (p ≤ 0.05) reduced Salmonella as compared to the untreated controls (~1.92-7.61 log CFU ml-1) while heat shock significantly (p ≤ 0.05) reduced Salmonella as compared to the untreated controls (~5.80-7.36 log CFU ml-1). Survival of Salmonella was higher in lamb juices than other juices. The fat content in lamb FL (3.25%) was significantly higher (p ≤ 0.05) than beef (1.30%) and goat FL (1.42%). Iron content in lamb FLM (~127 mg kg-1) was significantly (p ≤ 0.05) lower than beef (~233 mg kg-1) and goat FLM (~210 mg kg-1). The omega 6 and linoleic acid content in goat FLM (~36.0% and ~34.4%) was significantly higher (p ≤ 0.05) than beef (~29.1% and ~27.1%). Fat, fatty acids and iron may differentially protect Salmonella against the effect of heat in these juices.

Survival of Salmonella on Red Meat in Response to Dry Heat.

ABSTRACT: Red meat is associated with Salmonella outbreaks, resulting in negative impacts for the processing industry. Little work has been reported on the use of dry heat as opposed to moist heat against Salmonella on red meat. We determined the effect of drying at 25°C and dry heat at 70°C with ∼10% relative humidity for 1 h against 11 Salmonella strains of multiple serovars on beef, lamb, and goat and rubber as an inert surface. Each strain at ∼108 CFU/mL was inoculated (100 µL) onto ±1 g (1 cm2) of each surface and allowed to attach for 15 min in a microcentrifuge tube. Samples were then exposed to 70 and 25°C with ∼10% relative humidity in a heating block. Surviving Salmonella numbers on surfaces were enumerated on a thin agar layer medium. If numbers were below the limit of detection (2.01 log CFU/cm2), Salmonella cells were enriched before plating to determine the presence of viable cells. Water loss (percent) from meat after at 25 and 70°C was determined. Whole genomes of Salmonella were interrogated to identify the presence-absence of stress response genes (n = 30) related to dry heat that may contribute to the survival of Salmonella. The survival of Salmonella at 25°C was significantly higher across all surfaces (∼6.09 to 7.91 log CFU/cm2) than at 70°C (∼3.66 to 6.33 log CFU/cm2). On rubber, numbers of Salmonella were less than the limit of detection at 70°C. Water loss at 70°C (∼17.72 to 19.89%) was significantly higher than at 25°C (∼2.98 to 4.11%). Salmonella cells were not detected on rubber, whereas survival occurred on all red meat at 70°C, suggesting its protective effect against the effect of heat. All Salmonella strains carried 30 stress response genes that likely contributed to survival. A multi-antibiotic-resistant Salmonella Typhimurium 2470 exhibited an increase in heat resistance at 70°C on beef and lamb compared with other strains. Our work shows that dry heat at 70°C for 1 h against Salmonella on red meat is not a practical approach for effectively reducing or eliminating them from red meat.

Draft Genome Sequences of Four Antibiotic-Resistant Salmonella Strains Isolated from Australian Red Meat Animal Species.

The genome sequences of four antibiotic-resistant Salmonella strains isolated from red meat animals in Australia are presented. Multidrug-resistant Salmonella enterica serovar Heidelberg 329 and Salmonella enterica serovar Typhimurium 2470 harbored an IncHI2 plasmid similar to the multidrug-resistant S. Heidelberg strain N13-01290 plasmid pN13-01290_23 previously isolated in Canada.

Transfer of Campylobacter and Salmonella from Poultry Meat onto Poultry Preparation Surfaces.

Thermophilic Campylobacter and Salmonella enterica are major causes of gastrointestinal foodborne infection. Survival of these pathogens on food-associated surfaces is a risk contributing to their spread through the food system. This study examined the transfer of two strains each of C. jejuni, C. coli, Salmonella Enteritidis, and Salmonella Typhimurium from chicken meat to a knife or scissors used on either a plastic or wooden cutting board. Each strain of Campylobacter and Salmonella at ∼108 CFU mL-1 was inoculated (5 mL) onto 25 g of chicken meat with skin and allowed to attach (for 10 min). The meat was then cut (20 times per implement) into 1-cm2 pieces with either a knife or scissors on either a plastic or wooden cutting board. The numbers of pathogens transferred from meat onto cutting implements and cutting board surfaces were enumerated. The surfaces were subsequently either rinsed with water or rinsed with water and wiped with a kitchen towel to mimic commonly used superficial cleaning practices for these implements, and the numbers of pathogens were enumerated again. The bacterial numbers for both pathogens were determined on thin-layer agar. The attachment of the Salmonella strains to chicken meat (∼7.0 to 7.8 log CFU cm-2) was higher than the attachment of the Campylobacter strains (∼4.6 to 6.6 log CFU cm-2). All four Salmonella strains transferred in higher numbers (∼1.9 to 6.3 log CFU cm-2) to all surfaces than did the Campylobacter strains (∼1.1 to 3.9 log CFU cm-2). The transfer rates of both pathogens from the chicken meat to all the surfaces examined varied substantially between ∼0 and 21.1%. The highest rate of transfer (∼21.1%) observed was for C. coli 2875 when transferred from the chicken meat to the scissors. Most cleaning treatments reduced the numbers of both pathogens (∼0.3 to 4.1 log CFU cm-2) transferred to all the surfaces. Our study gives insights into the risks associated with the transfer of Campylobacter and Salmonella from poultry to the surfaces used in poultry preparation.

Trisodium phosphate and sodium hypochlorite are more effective as antimicrobials against Campylobacter and Salmonella on duck as compared to chicken meat.

Little work has been reported on the use of commercial antimicrobials against foodborne pathogens on duck meat. We investigated the effectiveness of trisodium phosphate (TSP) and sodium hypochlorite (SH) as antimicrobial treatments against Campylobacter and Salmonella on duck meat under simulated commercial water chilling conditions. The results were compared to the same treatments on well-studied chicken meat. A six strain Campylobacter or Salmonella cocktail was inoculated (5 ml) at two dilution levels (10(4) and 10(8) cfu/ml) onto 25 g duck or chicken meat with skin and allowed to attach for 10 min. The meat was exposed to three concentrations of pH adjusted TSP (8, 10 and 12% (w/v), pH 11.5) or SH (40, 50 and 60 ppm, pH 5.5) in 30 ml water under simulated spin chiller conditions (4 °C, agitation) for 10 min. In a parallel experiment the meat was placed in the antimicrobial treatments before inoculation and bacterial cocktails were added to the meat after the antimicrobial solution was removed while all other parameters were maintained. Untreated controls and controls using water were included in all experiments. Bacterial numbers were determined on Campylobacter blood-free selective agar and Mueller Hinton agar or xylose deoxycholate agar and tryptone soya agar using the thin agar layer method for Campylobacter and Salmonella, respectively. All TSP concentrations significantly (p<0.05) reduced numbers of Campylobacter (~1.2-6.4 log cfu/cm(2)) and Salmonella (~0.4-6.6 log cfu/cm(2)) on both duck and chicken meat. On duck meat, numbers of Campylobacter were less than the limit of detection at higher concentrations of TSP and numbers of Salmonella were less than the limit of detection at all concentrations of TSP except one. On chicken meat, numbers of Campylobacter and Salmonella were less than the limit of detection only at the lower inoculum level and higher TSP concentrations. By contrast only some of the concentrations of SH significantly (p<0.05) reduced numbers of Campylobacter and Salmonella (~0.2-1.5 log cfu/cm(2)) on both duck and chicken meats. None of the SH treatments resulted in numbers of either pathogen being less than limit of detection. Results indicate that chicken meat has the ability to effectively protect Campylobacter and Salmonella against the impact of trisodium phosphate and sodium hypochlorite while duck meat does not. This study suggests that trisodium phosphate has a strong potential for application in a commercial poultry processing to reduce Campylobacter and Salmonella specifically on duck meat.

Antimicrobial activity of gallic acid against thermophilic Campylobacter is strain specific and associated with a loss of calcium ions.

Gallic acid has been suggested as a potential antimicrobial for the control of Campylobacter but its effectiveness is poorly studied. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of gallic acid against Campylobacter jejuni (n = 8) and Campylobacter coli (n = 4) strains was determined. Gallic acid inhibited the growth of five C. jejuni strains and three C. coli strains (MIC: 15.63-250 µg mL(-1)). Gallic acid was only bactericidal to two C. coli strains (MBC: 125 and 62.5 µg mL(-1)). The mechanism of the bactericidal effect against these two strains (and selected non-susceptible controls) was investigated by determining decimal reduction times and by monitoring the loss of cellular content and calcium ions, and changes in cell morphology. Gallic acid did not result in a loss of cellular content or morphological changes in the susceptible strains as compared to the controls. Gallic acid resulted in a loss of calcium ions (0.58-1.53 µg mL(-1) and 0.54-1.17 µg mL(-1), respectively, over a 180 min period) from the susceptible strains but not the controls. Gallic acid is unlikely to be an effective antimicrobial against Campylobacter in a practical sense unless further interventions to ensure an effective bactericidal mode of action against all strains are developed.