Surface conditioning with bacteriophages reduces biofilm formation of Salmonella Heidelberg.

Salmonella remains one of the most common foodborne pathogens worldwide, and its resistance to antimicrobials has increased considerably over the years. In this context, was evaluated the action of three bacteriophages isolated or combined in inhibiting the adhesion and removal of Salmonella Heidelberg biofilm on a polystyrene surface. The bacteriophages UPF_BP1, UPF_BP2, UPF_BP3 and a pool of them were used for biocontrol of Salmonella Heidelberg biofilms on polystyrene surface in the action times of 3, 6 and 9 h. Individual and combined phages exhibited reductions in Salmonella Heidelberg adhesion of up to 83.4% and up to 64.0% in removal of preformed biofilm. The use of synergistic combinations between the phages is the most indicated option due to its potential to reduce biofilms. The use of the bacteriophage pool in surface conditioning is an alternative in the control of Salmonella Heidelberg biofilms.

Bacteriophages as an alternative for biological control of biofilm-forming Salmonella enterica.

Salmonellosis is one of the most common foodborne diseases worldwide. Surface adherence and biofilm formation are among the main strategies evolved by Salmonella to survive under harsh conditions and are risk factors for its spread through the food chain. Owing to the increase in antimicrobial resistance, there is a growing need to develop other methods to control foodborne pathogens, and bacteriophages have been suggested as a potential alternative for this purpose. The aim of this study was to evaluate bacteriophages as a biological control of Salmonella enterica serotypes to inhibit and remove bacterial biofilms. A total of 12 S. enterica isolates were selected for this study, all of which were biofilm producers. Seven bacteriophages were tested, individually and in a cocktail, for their host range and efficiency of plating (EOP). The phage cocktail was evaluated for its antibiofilm effect against the Salmonella biofilms. Phages UPF_BP1, UPF_BP2, UPF_BP3, UPF_BP6, and 10:2 possessed a broad lytic spectrum and could infect all S. enterica strains. Phages 10:2, UPF_BP6, and UPF_BP3 had high EOP in 10, 9, and 9 out of the 12 S. enterica strains, respectively. The cocktail was able to infect all S. enterica strains and had a high EOP in 10 out of 12 S. enterica isolates, presenting a broader host range than any of the tested single phages. A wide variation of inhibition among strains was observed, ranging from 14.72% to 88.53%. Multidrug-resistant and strong biofilm producer strains showed high biofilm inhibition levels by phage cocktail. Our findings demonstrate the ability of the cocktail to prevent biofilm formation and remove formed biofilms of Salmonella. These results indicate that the phage cocktail is a promising candidate to be used as an alternative for the control of Salmonella biofilms through surface conditioning.

Characterization of novel of temperate phages of Staphylococcus aureus isolated from bovine milk.

Bovine mastitis is an important disease in dairy cows, and Staphylococcus aureus is the most prevalent microorganism. Bacteriophages are considered an alternative to treat bacterial infections due to antimicrobial resistance crisis. In this study, we isolated and characterized novel S. aureus temperate phages, namely B_UFSM4 and B_UFSM5, from bovine milk. The complete genomes of B_UFSM4 and B_UFSM5 have 41.396 bp and 41.829 bp, respectively. The viruses have double-stranded DNA and linear architecture. Phylogenic similarity was observed by proteome with Staphylococcus phage phiPV83, CN125 and JS01. Therefore, the phages were classified into the family Siphoviridae, genus Biseptimavirus and order Caudovirales. In the host range, the B_UFSM4 and B_UFSM5 had lytic activity of 45.8% and 54.16%, respectively, inclusive on isolates from Staphylococcus sciuri and Rothia terrae. Thus, in this study, species novel of S. aureus temperate phages was isolated and characterized, these phages reveal similarities to each other; however, they are distinct from other species of S. aureus phages of the family Siphoviridae.

Genomic characterization of multidrug-resistant Salmonella Heidelberg E2 strain isolated from chicken carcass in southern Brazil.

Salmonella Heidelberg is a clinically-important serovar linked to food-borne illness, and commonly isolated from poultry products. Since 1962, Salmonella Heidelberg has been widely reported from poultry production systems in several countries, including Brazil. The emergence of multidrug-resistant (MDR) Salmonella Heidelberg strains in food animals underscores a significant food safety hazard. In our study, we performed antimicrobial susceptibility testing (AST) and Whole-genome sequencing (WGS) to identify the antimicrobial resistance (AMR) genes, pathogenicity mechanisms and virulence factors (VF) in Salmonella Heidelberg E2 strain recovered from a chicken carcass in Southern Brazil. Salmonella Heidelberg strain belonged to ST15 and showed to be susceptible to colistin (MIC ≤2 µg/mL) and multidrug-resistant to amoxicillin-clavulanic acid, gentamicin, ampicillin, cefaclor, cefazolin, ceftiofur, nalidixic acid, azithromycin, erythromycin, doxycycline, tetracycline and sulfonamide. We identified AMR genes mediating resistance to aminoglycosides (aac(6')-Iaa, aac(3)-VIa, aph(3')-Ia, aadA, 16S rrsD), ß-lactams (blaCTX-M-2), quinolones (parC), macrolides (acrB), tetracyclines (tet(A)), fosfomycin (fosA7) and sulfonamide (sul1). Interestingly, the mutation in parC T255S has never been reported among Salmonella Heidelberg strains. To our knowledge, this is the first report of a Salmonella enterica strain harbouring 16S rrsD 471G > A, acrB F28L and acrB L40P chromosomal point mutations. Three plasmid replicon types, ST2-IncHI2, ST2-IncHI2A and IncX1 were identified. Nine Salmonella Pathogenicity Islands and 98 virulence genes encoding virulence factors were identified associated with cell adhesion, invasion, intracellular survival and resistance to antimicrobial peptides. Although Salmonella Heidelberg E2 strain likely originated from poultry, cross-contamination during meat processing cannot be excluded. This study adds to our understanding of Salmonella Heidelberg transmission along the food-chain and informs ongoing regulatory discussions on Salmonella Heidelberg in poultry.

Salmonella Enteritidis forms biofilm under low temperatures on different food industry surfaces / Salmonella Enteritidis forma biofilme sob baixas temperaturas em diferentes superfícies da indústria de alimentos

ABSTRACT: We evaluated the influence of temperature on the ability of Salmonella Enteritidis (SE) to form biofilms on stainless steel, polyethylene, and polyurethane surfaces under different hygiene procedures. These materials were placed on SE culture and incubated at 42±1 ºC, 36±1 ºC, 25±1 ºC, 9±1 ºC, and 3±1 ºC for 4, 8, 12, and 24 h. Hot water at 45 ºC and 85 ºC, 0.5% peracetic acid solution, and 1% quaternary ammonia were used for hygienization. Biofilm formation occurred at all temperatures evaluated, highlighting at 3 ºC which has not been reported as an ideal temperature for the adhesion of SE to these materials. The SE adhered more often to polyethylene surfaces than to polyurethane and stainless steel surfaces (P<0.05). Peracetic acid and water at 85 ºC had similar hygienization efficiency (P<0.05) followed by quaternary ammonia whereas water at 45 ºC was not effective. SE adhered to these materials under low temperatures which to date have been deemed safe for food preservation.

RESUMO: Avaliou-se o efeito da temperatura na capacidade de Salmonella Enteritidis (SE) formar biofilme em superfícies de aço inoxidável, polietileno e poliuretano e diferentes processos de higienização. Corpos de prova destes materiais foram postos frente a culturas de SE e incubados a 42±1 ºC, 36±1 ºC, 25±1 ºC, 9±1 ºC e 3±1 ºC por 4, 8, 12 e 24 horas. Para a higienização foram testados água aquecida a 45ºC e 85 ºC e soluções de ácido peracético 0,5% e amônia quaternária 1%. Verificou-se a formação de biofilmes em todas as temperaturas avaliadas, ressaltando-se a 3 ºC, ainda não citada como propícia para adesão de SE. Houve maior adesão ao polietileno do que ao poliuretano e ao aço inoxidável (P<0.05). Para higienização, o ácido peracético e a água a 85 ºC tiveram ação semelhante (P<0.05), seguidos por amônia quaternária, enquanto que a água a 45 ºC não foi eficaz. Todos os materiais avaliados propiciaram a aderência de SE, mesmo sob temperaturas baixas, consideradas até então seguras para a conservação dos alimentos.

Responsiveness of the interrenal tissue of Jundiá (Rhamdia quelen) to an in vivo ACTH test following acute exposure to sublethal concentrations of agrichemicals.

As in many aquatic environments, pollution is a widespread problem in Southern Brazil. In our previous work, we demonstrated that sublethal contamination with some agrichemicals impairs the capacity of fishes to elevate cortisol levels in response to an additional acute stressor. In earlier experiments, the experimental design did not allow us to conclude where this effect occurs. In the present work, we used the adrenocorticotropic hormone (ACTH) challenge test to help us identify if the impairment occur in the interrenal tissue. For this purpose, five experiments were conducted, each with one specific agrichemical (methyl-parathion, atrazine+simazine, atrazine, tebuconazole, and glyphosate) in sublethal concentrations of 16.6% of the LC(50-96h), as previously determined. Fish were subjected to the ACTH challenge test protocol as follows: group 1, were non-injected and maintained as the specific control group; group 2 received an injection of the vehicle alone (the saline group); and group 3 receive an injection of ACTH. One hour later, blood samples were taken from the caudal plexus, using sterile syringes. In all specific control groups, the injection of ACTH induced a strong rise in plasma cortisol, compared with the fish injected only with the vehicle and the non-injected group. Fish exposed to methyl-parathion and tebuconazole did not elevate cortisol in response to the ACTH injection, with values significantly lower than the control fish. Fish exposed to sublethal concentrations of atrazine+simazine, atrazine, and glyphosate showed a rise in plasma cortisol very similar to the control fish. We conclude that the ACTH challenge test revealed that R. quelen exposed to sublethal concentrations of tebuconazole and methyl-parathion had a reduced ability to elevate plasma cortisol in response to an intraperitoneal (i.p.) injection of exogenous ACTH, indicating that the interrenal tissue is the site of the impairment within the HPI axis. These ACTH challenge tests also revealed that the impairment of the cortisol response verified in fish exposed to atrazine+simazine and glyphosate, as shown in our previous work, seems to be related to steps of cortisol secretion in higher levels within the HPI axis.

Cortisol response to acute stress in jundiá Rhamdia quelen acutely exposed to sub-lethal concentrations of agrichemicals.

Exposure to agrichemicals can have deleterious effects on fish, such as disruption of the hypothalamus-pituitary-inter-renal axis (HPI) that could impair the ability of fish to respond to stressors. In this study, fingerlings of the teleost jundiá (Rhamdia quelen) were used to investigate the effects of the commonly used agrichemicals on the fish response to stress. Five common agrichemicals were tested: the fungicide - tebuconazole, the insecticide - methyl-parathion, and the herbicides - atrazine, atrazine+simazine, and glyphosate. Control fishes were not exposed to agrichemicals and standard stressors. In treatments 2-4, the fishes were exposed to sub-lethal concentrations (16.6%, 33.3%, and 50% of the LC(50)) of each agrichemical for 96 h, and at the end of this period, were subjected to an acute stress-handling stimulus by chasing them with a pen net. In treatments 5-7 (16.6%, 33.3%, and 50% of the LC(50)), the fishes were exposed to the same concentrations of the agrichemicals without stress stimulus. Treatment 8 consisted of jundiás not exposed to agrichemicals, but was subjected to an acute stress-handling stimulus. Jundiás exposed to methyl-parathion, atrazine+simazine, and glyphosate presented a decreased capacity in exhibiting an adequate response to cope with stress and in maintaining the homeostasis, with cortisol level lower than that in the control fish (P<0.01). In conclusion, the results of this study clearly demonstrate that the acute exposure to sub-lethal concentrations of methyl-parathion, atrazine+simazine, and glyphosate exert a deleterious effect on the cortisol response to an additional acute stressor in the jundiá fingerlings.