As antimicrobial resistance continues to escalate, the exploration of alternative approaches to safeguard food safety becomes more crucial than ever. Phage endolysins are enzymes derived from phages that possess the ability to break down bacterial cell walls. They have emerged as promising antibacterial agents suitable for integration into food processing systems. Their application as food preservatives can effectively regulate pathogens, thus contributing to an overall improvement in food safety. This review summarizes the latest techniques considering endolysins’ potential for food safety. These techniques include native and engineered endolysins for controlling bacterial contamination at different points within the food production chain. However, we find that characterizing endolysins through in vitro methods proves to be time consuming and resource intensive. Alternatively, the emergence of advanced high-throughput sequencing technology necessitates the creation of a robust computational framework to efficiently characterize recently identified endolysins, paving the way for future research. Machine learning encompasses potent tools capable of analyzing intricate datasets and pattern recognition. This study briefly reviewed the use of these industry 4.0 technologies for advancing the research in food industry. We aimed to provide current status of endolysins in food industry and new insights by implementing these industry 4.0 strategies revolutionizes endolysin development. It will enhance food safety, customization, efficiency, transparency, and collaboration while reducing regulatory hurdles and ensuring timely product availability.

Salmonella spp. are one of the most important foodborne bacterial pathogens in human beings and animals. This study aimed to analyze the prevalence and characterization of Salmonella from broilers in Shandong, China. A total of 67 Salmonella were recovered from 600 rectal swabs collected from 3 large-scale intensive broiler farms (67/600, 11.2%) between May and October 2018. Among Salmonella isolates, the most common serovars were S. enteritidis and S. typhimurium. The highest occurrence of resistance observed was for polymyxin (100%), followed by ampicillin (68.7%). The multidrug-resistant Salmonella isolation rate was observed to be 53.7%. Four β-lactamase genes were detected among the isolates, and all the isolates carried blaTEM (67/67, 100%), followed by blaOXA (19/67, 28.4%), blaCTX-M (17/67, 25.4%), and blaPSE (7/67, 10.4%). Four plasmid-mediated quinolone resistance gene were detected among the isolates; the prevalent resistance genes was aac(6')-Ib-cr (18/67, 26.9%), followed by oqxB (9/67, 13.4%), qnrB (6/67, 9.0%), and qnrD (1/67, 1.5%). The prevalent rate of mcr-1 was 6.0% (4/67). Class 1 integrons were detected in 26.9% of these isolates and contained 7 groups of resistance gene cassettes. Multilocus sequence typing analysis revealed 7 sequence types, and ST11 was the most frequent sequence types. This study indicated that reduction of Salmonella and strict control on the use of antibiotics in more than 5,000 million broilers in Shandong are the vitally important measures to keep public health.

Clostridium perfringens is an important foodborne pathogen that can have severe consequences, including mortality and economic losses. In this study, the gene encoding cpp-lys, an endolysin from the C. perfringens phage cpp has been cloned and overexpressed. The encoded protein was characterized, and then its efficacy in controlling C. perfringens on lettuce was evaluated. The endolysin cpp-lys presented lytic activity against seven strains of C. perfringens that produce different types of toxins. It maintained stability across a wide range of temperatures (4 °C - 50 °C), and demonstrated tolerance to varying pH levels (4-9). Storage of endolysin cpp-lys under room-temperature conditions (16 °C-25 °C) and cold-temperature conditions (4 °C, -20 °C, and -80 °C) for 30 days did not affect its lytic activity. However, the lytic activity of cpp-lys decreased by 40 % and 18 % after storage for 30 d at 42 °C and 37 °C, respectively. The endolysin cpp-lys did not display cytotoxic activity against normal eukaryotic cells. The bacterial viability on lettuce was significantly lower in the group treated with endolysin cpp-lys than in the PBS group, and >4-log of C. perfringens J1 were removed within 15 min. Cpp-lys plus Zn²⁺ inhibited the activity of cpp-lys. The EDTA-treated cpp-lys significantly reduced the number of bacteria by up to 0.6-log CFU compared with the endolysin cpp-lys group. The findings of this study demonstrated that endolysin cpp-lys has potential applications in controlling C. perfringens in the food industry.

Waterfowl have a high likelihood of being infected with Riemerella anatipestifer. Although the pathogen is found in domestic ducks, turkeys, geese, and wild birds, there is little information available about the consequences of infection during egg laying and hatching in chickens. Here, we present the first report of a novel sequence type of R. anatipestifer S63 isolated from chickens in China. On the basis of pan-genome analysis, we showed S63's genome occupies a distinct branch with other R. anatipestifer isolates from other hosts. Galleria mellonella larval tests indicated that S63 is less virulent than R. anatipestifer Ra36 isolated from ducks. Ducks and hens are susceptible to S63 infection. There is no mortality rate for chickens or ducks, but adult chickens experience neurological symptoms that reduce egg production and hatching rates. In chickens, S63 might be passed vertically from parents to offspring, resulting in "jelly-like" lifeless embryos. Using quantitative PCR, S63 was detected in the brain, liver, reproductive organs, and embryos. As far as we know, this is the first report of R. anatipestifer in hens, a disease that can reduce egg productivity, lower hatching rates, and produce jelly-like lifeless embryos, and the first report to raise the possibility that hens can be infected by roosters via semen.

Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB_SalS_JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB_SalS_JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB_SalS_JNS02 exhibited remarkable stability across a wide pH range (pH 3-12) and temperatures ranging from 30 to 80°C. Genome sequencing analysis provided valuable insights into the genetic composition of vB_SalS_JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB_SalS_JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100, 70, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB_SalS_JNS02 (10⁸ PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB_SalS_JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry.