Screening and Genomic Profiling of Antimicrobial Bacteria Sourced from Poultry Slaughterhouse Effluents: Bacteriocin Production and Safety Evaluation

Abstract

Background/Objectives: Antimicrobial-resistant (AMR) pathogens represent a serious threat to public health, particularly in food production systems where antibiotic use remains widespread. As a result, alternative antimicrobial treatments to antibiotics are essential for effectively managing bacterial infections. This study aimed to identify and characterize novel antimicrobial peptides produced by bacteria, known as bacteriocins, as well as to recognize safe bacteriocin-producing strains, sourced from poultry slaughterhouse effluents. Methods: A total of 864 bacterial isolates were collected across eight stages of a poultry slaughter line and screened for antimicrobial activity against Gram-positive and Gram-negative indicator strains. Whole-genome sequencing (WGS) was performed on 12 selected strains, including Enterococcus faecium (6 isolates), Lactococcus lactis (1 isolate), Lactococcus garvieae (1 isolate) and Escherichia coli (4 isolates). The presence of bacteriocin gene clusters (BGC), antibiotic resistance genes (ARG), and virulence factors (VF) was analyzed. The antimicrobial activity of a novel bacteriocin was further evaluated using in vitro cell-free protein synthesis (IV-CFPS). Results: WGS revealed multiple BGCs, including a novel class IId bacteriocin, lactococcin P1A (LcnP1A), in L. lactis SWD9. LcnP1A showed antimicrobial activity against various indicator strains, including Listeria monocytogenes. While most bacteriocin-encoding strains harbored ARGs and VFs, E. faecium SWG6 was notable for its absence of ARGs and minimal VFs, highlighting its potential as a probiotic. Conclusions: These findings underscore the importance of discovering novel bacteriocins and safer bacteriocin producing strains to address antimicrobial resistance in the food chain. Further research would validate the efficacy of both the novel lactococcin P1A bacteriocin and the E. faecium SWG6 isolate for application in processed food and animal production systems.

Keywords: E. coli; Enterococcus faecium; Lactococcus garvieae; Lactococcus lactis; bacteriocins; colicin; enterocins; lactococcins; microcin.

Figure 1

Schematic representation of the various locations from which water samples were collected in the poultry slaughterhouse. The numbers shown in the graph indicate the total isolates active against P. damnosus CECT4797. The figures in parentheses represent the number of bacteria subsequently selected for evaluation of their antimicrobial activity by using the stamp-on-agar test (STOAT). The asterisk denotes the isolates that showed activity against E. coli DH5α, with the numbers in parentheses indicating the isolates selected for their highest antimicrobial activity. The figures in the central square represent the total number of bacteria chosen for further analysis from all water samples.

Figure 2

(A) Amino acid sequence alignment of the putative bacteriocins encoded by E. faecium SCH10 (EnkA-, EnkZ-, and EnkD-like bacteriocins), E. faecium SWG6 (EntP-like bacteriocin), and L. garvieae SWE11 (GarQ-like bacteriocin). The leader sequences are underlined. An asterisk (*) indicates a single fully conserved residue, a colon (:) indicates conservation within groups of residues with strongly similar properties, and a period (.) indicates conservation within groups of residues with weakly similar properties. (B) Genetic organization of the hypothetical bacteriocin gene cluster (BGC) identified in E. faecium SWE11, which includes genes encoding EnkA-, EnkZ-, and EnkD-like bacteriocins, alongside the genetic organization of the bacteriocin gene cluster from E. faecium NKR-3-5.

Figure 3

(A) Genetic organization of the three hypothetical bacteriocin gene clusters (BGC) identified in the L. lactis SWD9 genome. The structural genes for putative lactococcins are indicated by green arrows, while immunity genes are shown with red arrows. The genes lcnP1C and lcnP1D encode a putative ABC transporter and a putative transport accessory protein, respectively. (B) Amino acid sequence alignment of the putative bacteriocins LcnP1A, LcnP2A, LcnP3A, and LcnP4A with garvicin Q (GarQ), lactococcin A (LcnA), and lactococcin B (LcnB). The leader sequences are underlined. An asterisk (*) indicates a single fully conserved residue, a colon (:) indicates conservation within groups of residues with strongly similar properties, and a period (.) indicates conservation within groups of residues with weakly similar properties. (C). Antimicrobial activity against P. damnosus CECT 4797 and (D) L. monocytogenes CECT 4032 was evaluated using a spot-on-agar test (SOAT). This involved application to the surfaces of agar plates overlaid with a culture of the indicator microorganisms 5 µL of LcnP1A, LcnP2A, LcnP3A, and LcnP4A synthesized and produced individually by an IV-CFPS procedure, or 2 µL of chemically synthetized GarQ, LcnA, and LcnB.