Non-Antibiotics Strategies to Control Salmonella Infection in Poultry

Salmonella spp. is a facultative intracellular pathogen causing localized or systemic infections, involving economic and public health significance, and remains the leading pathogen of food safety concern worldwide, with poultry being the primary transmission vector. Antibiotics have been the main strategy for Salmonella control for many years, which has allowed producers to improve the growth and health of food-producing animals. However, the utilization of antibiotics has been reconsidered since bacterial pathogens have established and shared a variety of antibiotic resistance mechanisms that can quickly increase within microbial communities. The use of alternatives to antibiotics has been recommended and successfully applied in many countries, leading to the core aim of this review, focused on (1) describing the importance of Salmonella infection in poultry and the effects associated with the use of antibiotics for disease control; (2) discussing the use of feeding-based (prebiotics, probiotics, bacterial subproducts, phytobiotics) and non-feeding-based (bacteriophages, in ovo injection, vaccines) strategies in poultry production for Salmonella control; and (3) exploring the use of complementary strategies, highlighting those based on -omics tools, to assess the effects of using the available antibiotic-free alternatives and their role in lowering dependency on the existing antimicrobial substances to manage bacterial infections in poultry effectively.

Lactobacillus reuteri and Enterococcus faecium from Poultry Gut Reduce Mucin Adhesion and Biofilm Formation of Cephalosporin and Fluoroquinolone-Resistant Salmonella enterica

Non-typhoidal Salmonella (NTS) can cause infection in poultry, livestock, and humans. Although the use of antimicrobials as feed additives is prohibited, the previous indiscriminate use and poor regulatory oversight in some parts of the world have resulted in increased bacterial resistance to antimicrobials, including cephalosporins and fluoroquinolones, which are among the limited treatment options available against NTS. This study aimed to isolate potential probiotic lactic acid bacteria (LAB) strains from the poultry gut to inhibit fluoroquinolone and cephalosporin resistant MDR Salmonella Typhimurium and S. Enteritidis. The safety profile of the LAB isolates was evaluated for the hemolytic activity, DNase activity, and antibiotic resistance. Based on the safety results, three possible probiotic LAB candidates for in vitro Salmonella control were chosen. Candidate LAB isolates were identified by 16S rDNA sequencing as Lactobacillus reuteri PFS4, Enterococcus faecium PFS13, and Enterococcus faecium PFS14. These strains demonstrated a good tolerance to gastrointestinal-related stresses, including gastric acid, bile, lysozyme, and phenol. In addition, the isolates that were able to auto aggregate had the ability to co-aggregate with MDR S. Typhimurium and S. Enteritidis. Furthermore, LAB strains competitively reduced the adhesion of pathogens to porcine mucin Type III in co-culture studies. The probiotic combination of the selected LAB isolates inhibited the biofilm formation of S. Typhimurium FML15 and S. Enteritidis FML18 by 90% and 92%, respectively. In addition, the cell-free supernatant (CFS) of the LAB culture significantly reduced the growth of Salmonella in vitro. Thus, L. reuteri PFS4, E. faecium PFS13, and E. faecium PFS 14 are potential probiotics that could be used to control MDR S. Typhimurium and S. Enteritidis in poultry. Future investigations are required to elucidate the in vivo potential of these probiotic candidates as Salmonella control agents in poultry and animal feed.

Opposing roles of Granzymes A and B in the immune response to intestinal infection

The cytotoxic proteases Granzymes A (GzmA) and B (GzmB) are key components of the arsenal used by cytotoxic immune cells to kill virally infected/damaged cells. Until now, there has been no evidence that GzmA/B proteins contribute to combating intracellular bacterial pathogens in mammals. Here, we find that the route of infection of the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium reveals the distinct roles that Granzymes play in defending against bacterial infection. We used Gzma-/-Gzmb-/- mice to discover that Granzymes are required to protect mice against oral infection with Salmonella. However, Granzymes do not play a role in systemic infection. We investigated the tissue-specific expression of Granzymes and determined that intestinal intraepithelial lymphocytes (IEL) are the only cell types that express Granzymes in healthy non-infected mice. In fact, IEL are essential and sufficient for the protective effects of Granzymes against Salmonella infection. Intriguingly, we found that GzmA and GzmB play opposing roles in Salmonella control, with GzmA being protective against infection whilst GzmB promoted infection. Both GzmA and GzmB proteins functioned independently of the pore-forming molecule Perforin. Our study reveals that IEL-expressed Granzymes play significant and distinct functions in host defense from oral bacterial infection.

Using Risk-Benefit Analysis for Controlling Salmonella in Chicken Meat

We have created a risk-benefit analysis (RBA) model to assist in food safety decision-making by analyzing Salmonella control in Brazilian chicken meat. First, we described the issues in a risk profile and used a 5 × 5 matrix to rank the risks associated with Salmonella. We then classified the magnitude of benefits and costs of control measures using another matrix. Finally, we verified the beneficial effects of recommended control measures using Quantitative Microbiological Risk Assessment (QMRA). The RBA classified Salmonella contamination as risk 6, indicating that control measures should be taken in the short and medium terms. It also recommended the adoption of biosecurity measures on farms to reduce the prevalence of Salmonella in birds, better control of carcass washings and chiller tank management, and information placement on packages and campaigns to raise the awareness of the population about the need to control Salmonella contamination before consumption. On the other hand, it did not recommend better controls at scalding and defeathering. QMRA confirmed the beneficial effects of the recommended control measures. For example, as Salmonella prevalence in poultry increased from 4.04% to 50%, the risk of infection per serving also increased from 0.0080 to 0.071. Although better controls in washings and chiller tank management did not affect the risk of infection, it reduced Salmonella counts on carcasses. We assume that the presence of Salmonella on carcasses was due to improper thermal processing or cross contamination, which increased the risk from 0.0080 to 0.015962. The RBA demonstrated the logic involved in the adoption of control measures, and this can be helpful in the risk management of food safety issues.

Impact of Salmonella Control Programmes in Poultry on Human Salmonellosis Burden in Greece

Since 2008, veterinary authorities in Greece have implemented national control programmes (NSCPs) targeting S. Enteritidis (SE) and S. Typhimurium (ST) in poultry. We assessed the effect of the programs on the reported number of human isolates. Using monthly data for 2006–2017, we defined two groups (SE, ST) and one control group with serotypes unrelated to poultry or eggs. For SE we also analysed data for 2006–2015 due to a multi-county SE outbreak in 2016. We performed an interrupted time series analysis and used a negative binominal regression model. For both SE and ST, there was no significant trend of the isolation rate before or after NSCPs’ introduction. After the NSCPs’ introduction there was an increasing rate (IRR: 1.005, 95% CI: 1.001–1.008) for control serotypes and a decreasing one for SE (IRR: 0.990, 95% CI: 0.986–0.995) (for 2009 to 2015 analysis). From 2006 to 2017, NSCPs had a statistically significant impact on the number of SE isolates that decreased by 49% (IRR:0.511, 95% CI: 0.353–0.739). No impact was shown on the number of ST (p-value = 0.741) and control isolates (p = 0.069). As a conclusion, NSCP’s implementation was associated with decreased SE isolates and overall burden of salmonellosis; however further measures aiming at human salmonellosis due to ST, should be considered.

The Occurrence of Antimicrobial-Resistant Salmonella enterica in Hatcheries and Dissemination in an Integrated Broiler Chicken Operation in Korea

Positive identification rates of Salmonella enterica in hatcheries and upstream breeder farms were 16.4% (36/220) and 3.0% (6/200), respectively. Among the Salmonella serovars identified in the hatcheries, S. enterica ser. Albany (17/36, 47.2%) was the most prevalent, followed by the serovars S. enterica ser. Montevideo (11/36, 30.6%) and S. enterica ser. Senftenberg (5/36, 13.9%), which were also predominant. Thirty-six isolates showed resistance to at least one antimicrobial tested, of which 52.8% (n = 19) were multidrug resistant (MDR). Thirty-three isolates (enrofloxacin, MIC ≥ 0.25) showed point mutations in the gyrA and parC genes. One isolate, S. enterica ser. Virchow, carrying the blaCTX-M-15 gene from the breeder farm was ceftiofur resistant. Pulsed-field gel electrophoresis (PFGE) showed that 52.0% S. enterica ser. Montevideo and 29.6% S. enterica ser. Albany isolates sourced from the downstream of hatcheries along the broiler chicken supply chain carried the same PFGE types as those of the hatcheries. Thus, the hatcheries showed a high prevalence of Salmonella isolates with high antimicrobial resistance and no susceptible isolate. The AMR isolates from hatcheries originating from breeder farms could disseminate to the final retail market along the broiler chicken supply chain. The emergence of AMR Salmonella in hatcheries may be due to the horizontal spread of resistant isolates. Therefore, Salmonella control in hatcheries, particularly its horizontal transmission, is important.

Characterization of Salmonella Phage LPST153 That Effectively Targets Most Prevalent Salmonella Serovars

Foodborne diseases represent a major risk to public health worldwide. In this study, LPST153, a novel Salmonella lytic phage with halo (indicative of potential depolymerase activity) was isolated by employing Salmonella enterica serovar Typhimurium ATCC 13311 as the host and had excellent lytic potential against Salmonella. LPST153 is effectively able to lyse most prevalent tested serotypes of Salmonella, including S. Typhimurium, S. Enteritidis, S. Pullorum and S. Gallinarum. Morphological analysis revealed that phage LPST153 belongs to Podoviridae family and Caudovirales order and could completely prevent host bacterial growth within 9 h at multiplicity of infection (MOI) of 0.1, 1, 10 and 100. LPST153 had a latent period of 10 min and a burst size of 113 ± 8 PFU/cell. Characterization of the phage LPST153 revealed that it would be active and stable in some harsh environments or in different conditions of food processing and storage. After genome sequencing and phylogenetic analysis, it is confirmed that LPST153 is a new member of the Teseptimavirus genus of Autographivirinae subfamily. Further application experiments showed that this phage has potential in controlling Salmonella in milk and sausage. LPST153 was also able to inhibit the formation of biofilms and it had the ability to reduce and kill bacteria from inside, including existing biofilms. Therefore, the phage LPST153 could be used as a potential antibacterial agent for Salmonella control in the food industry.