Comparative evaluation of the effectiveness of alcohol-based sanitizers, UV-C radiation and hot air on three-age Salmonella biofilms.

Dry sanitation is recommended to control contamination and prevent microbial growth and biofilm formation in the low-moisture food manufacturing plants. The objective of this study was to evaluate the effectiveness of dry sanitation protocols on Salmonella three-age biofilms formed on stainless steel (SS) and polypropylene (PP). Biofilms were formed for 24, 48 and 96 h at 37 °C using a cocktail of six Salmonella strains (Muenster, Miami, Glostrup, Javiana, Oranienburg, Yoruba) isolated from the peanut supply chain. Then, the surfaces were exposed to UV-C radiation, hot air (90 °C), 70% ethanol and a commercial product based on isopropyl alcohol for 5, 10, 15 and 30 min. After 30min exposure, on PP the reductions ranged from 3.2 to 4.2 log CFU/cm2 for UV-C, from 2.6 to 3.0 log CFU/cm2 for hot air, from 1.6 to 3.2 log CFU/cm2 for 70% ethanol and from 1.5 to 1.9 log CFU/cm2 for the commercial product. On SS, after the same exposure time, reductions of 1.3-2.2 log CFU/cm2, 2.2 to 3.3 log CFU/cm2, 1.7 to 2.0 log CFU/cm2 and 1.6 to 2.4 log CFU/cm2 were observed for UV-C, hot air, 70% ethanol and commercial product, respectively. UV-C was the only treatment affected by the surface material (p < 0.05) whereas the biofilm age influenced the effectiveness of UV-C and hot air (p < 0.05). For most treatment, there was significant difference among the exposure times (p < 0.05). Overall, the fastest loss in the biofilm viability was noted in the first 5 min, followed by a tail phase. The time predicted by the Weibull model for the first decimal reduction ranged from 0.04 to 9.9 min on PP and from 0.7 to 8.5 min on SS. In addition, the Weibull model indicates that most of treatments (79%) required a long-term exposure time (>30 min) to achieve 3-log reductions of Salmonella biofilms. In summary, UV-C showed the best performance on PP whereas hot air was noted to be the most effective on SS.

Multidrug-Resistant Biofilms (MDR): Main Mechanisms of Tolerance and Resistance in the Food Supply Chain.

Biofilms are mono- or multispecies microbial communities enclosed in an extracellular matrix (EPS). They have high potential for dissemination and are difficult to remove. In addition, biofilms formed by multidrug-resistant strains (MDRs) are even more aggravated if we consider antimicrobial resistance (AMR) as an important public health issue. Quorum sensing (QS) and horizontal gene transfer (HGT) are mechanisms that significantly contribute to the recalcitrance (resistance and tolerance) of biofilms, making them more robust and resistant to conventional sanitation methods. These mechanisms coordinate different strategies involved in AMR, such as activation of a quiescent state of the cells, moderate increase in the expression of the efflux pump, decrease in the membrane potential, antimicrobial inactivation, and modification of the antimicrobial target and the architecture of the EPS matrix itself. There are few studies investigating the impact of the use of inhibitors on the mechanisms of recalcitrance and its impact on the microbiome. Therefore, more studies to elucidate the effect and applications of these methods in the food production chain and the possible combination with antimicrobials to establish new strategies to control MDR biofilms are needed.

Biofilm formation and resistance to sanitizers by Salmonella spp. Isolated from the peanut supply chain.

Pathogens such as Salmonella can be difficult to control in low-moisture food (LMF) processing plants and because of this its presence especially in biofilm mode should be prevented in such an environment. This study evaluated the capacity of Salmonellastrains isolated from the peanut supply chain (S. Muenster, S. Miami, S. Glostrup, S. Javiana, S. Oranienburg and S. Yoruba) to form biofilm as well as their resistance to sanitizers (sodium hypochlorite, peracetic acid, quaternary ammonium, alkaline chlorinated solution and biguanide). Regarding biofilm formation, there was no significant difference (p > 0.05) among the strains tested singly on AISI 304 stainless steel (SS) and polypropylene (PP) coupons at the same temperature. However, a difference (p < 0.05) between the temperatures was noted in the first hours of incubation. The sessile cells reached counts between 3 and 4 log CFU/cm2 at 25 °C whereas>5 log CFU/cm2 was observed at 37 °C after 8 h. From 24 h the counts were above 6 log CFU/cm2 for both temperatures. Nevertheless, the SEM images of the 6-strain pool showed the highest density of adhered cells after 48 h at 25 °C and 24 h at 37 °C on PP, and after 48 h at both temperatures on SS. Peracetic acid (300 mg/L) had the shortest action time against 24-h biofilm on SS and PP, with sessile cell counts below the limit of detection (0.59 log CFU/cm2) after 3 min. For 48-h and 96-h biofilm, sodium hypochlorite (200 mg/L) decreased>4 log CFU/cm2 within 5 min. Quaternary ammonium (350 mg/L) and chlorinated alkaline detergent (200 mg/L) showed intermediate performances. Only biguanide (800 mg/L) did not reduce the biofilm counts to below the limit of detection in any of the conditions evaluated. The results indicated high biofilm formation ability of the Salmonella strains isolated from the peanut supply chain. Nevertheless, in general the biofilms were sensitive to most sanitizers within 15 min of treatment. This was the first study which evaluated biofilm formation by Salmonella isolated from the peanut supply chain. Data obtained here will contribute to optimize the hygiene practices in LMF manufacturing plants.

Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee.

Some species from Aspergillus section Nigri are morphologically very similar and altogether have been called A. niger aggregate. Although the species included in this group are morphologically very similar, they differ in their ability to produce mycotoxins and other metabolites and their taxonomical status has evolved continuously. Among them, A. niger and A. welwitschiae are ochratoxin A and fumonisin B2 producers and their detection and/or identification is of crucial importance for food safety. The aim of this study was the development of a real-time PCR-based method for simultaneous discrimination of A. niger and A. welwitschiae from other species of the A. niger aggregate isolated from coffee beans. One primer pair and a hybridization probe specific for detection of A. niger and A. welwitschiae strains were designed based on the BenA gene sequences, and used in a Real-time PCR assay for the rapid discrimination between both these species from all others of the A. niger aggregate. The Real-time PCR assay was shown to be 100% efficient in discriminating the 73 isolates of A. niger/A. welwitschiae from the other A. niger aggregate species analyzed as a negative control. This result testifies to the use of this technique as a good tool in the rapid detection of these important toxigenic species.

Long-term kinetics of Salmonella Typhimurium ATCC 14028 survival on peanuts and peanut confectionery products.

Due to recent large outbreaks, peanuts have been considered a product of potential risk for Salmonella. Usually, peanut products show a low water activity (aw) and high fat content, which contribute to increasing the thermal resistance and survival of Salmonella. This study evaluated the long-term kinetics of Salmonella survival on different peanut products under storage at 28°C for 420 days. Samples of raw in-shell peanuts (aw = 0.29), roasted peanuts (aw = 0.39), unblanched peanut kernel (aw = 0.54), peanut brittle (aw = 0.30), paçoca (aw = 0.40) and pé-de-moça (aw = 0.68) were inoculated with Salmonella Typhimurium ATCC 14028 at two inoculum levels (3 and 6 log cfu/ g). The Salmonella behavior was influenced (p<0.05) by aw, lipid, carbohydrate and protein content. In most cases for both inoculum levels, the greatest reductions were seen after the first two weeks of storage, followed by a slower decline phase. The lowest reductions were verified in paçoca and roasted peanuts, with counts of 1.01 and 0.87 log cfu/ g at low inoculum level and 2.53 and 3.82 log cfu/ g at high inoculum level at the end of the storage time. The highest loss of viability was observed in pé-de-moça, with absence of Salmonella in 10-g after 180 days at low inoculum level. The Weibull model provided a suitable fit to the data (R2≥0.81), with δ value ranging from 0.06 to 49.75 days. Therefore, the results demonstrated that Salmonella survives longer in peanut products, beyond the shelf life (>420 days), especially in products with aw around 0.40.