Investigating the Potential of Natural Antimicrobial Molecules for Reducing Samonella enterica serovar Enteritidis Colonization in Chickens

Salmonella Enteritidis is a major pathogen that causes foodborne human illness in the United States. Despite several control measures adopted for reducing the pathogen by pre-harvest and post-harvest approaches, Salmonella is widespread in poultry leading to elevated incidence rates. Epidemiological studies suggest that poultry and poultry products are the major vehicles for transmission of S. Enteritidis to humans. Chickens serve as natural hosts for S. Enteritidis with the cecum being the principal site of colonization. Cecal colonization of S. Enteritidis in birds leads to the contamination of carcasses during slaughter, contamination of eggshell with feces, and contamination of egg yolk, albumen and shell membranes by transovarian route. Therefore, reducing S. Enteritidis carriage in chickens is critical for reducing human infections. In this Ph. D. dissertation, the efficacy of three natural molecules, caprylic acid (CA), a medium chain fatty acid, and trans-cinnamaldehyde (TC) and eugenol (EG), two phytophenols, were investigated for reducing cecal colonization and fecal shedding of S. Enteritidis in chickens. In addition, cell culture studies, quantitative real-time PCR and DNA microarray were used to elucidate the molecular mechanisms behind the antimicrobial action of TC and EG. Results revealed that prophylactic and therapeutic supplementation of CA, TC and EG in feed significantly reduced S. Enteritidis populations in the cecum and cloaca of treated chickens, compared to the control birds (P \u3c 0.05). Feeding of CA, TC or EG did not adversely affect the body weight, feed intake, pH, or endogenous cecal bacterial population in treated chickens, compared to the negative controls. Follow up studies revealed that the natural molecules reduced S. Enteritidis motility and invasion of avian intestinal epithelial cells, in vitro (P \u3c 0.05). Real-Time quantitative PCR results indicated that the expression of major virulence genes such as hilA, hilD, invF, flhC , and motA in S. Enteritidis was reduced significantly by CA, TC and EG (P \u3c 0.05). The DNA microarray data revealed that the TC and EG exerted antimicrobial effect on S. Enteritidis by multiple mechanisms, including down-regulation of genes responsible for flagellar motility, Salmonella Pathogenicity Island 1, type three secretion system, translocation and effector proteins, hyb virulence operon, outer membrane proteins, cell division, transcription, translation, metabolism, biosynthetic pathways, and terminal electron acceptors. The efficacy of CA, TC and EG in reducing S. Enteritidis in chickens could be utilized in the control of the pathogen in chickens as a pre-harvest treatment for potentially decreasing foodborne diseases caused by the pathogen.

Investigating the Potential of Natural Antimicrobial Molecules for Reducing Samonella enterica serovar Enteritidis Colonization in Chickens

Salmonella Enteritidis is a major pathogen that causes foodborne human illness in the United States. Despite several control measures adopted for reducing the pathogen by pre-harvest and post-harvest approaches, Salmonella is widespread in poultry leading to elevated incidence rates. Epidemiological studies suggest that poultry and poultry products are the major vehicles for transmission of S. Enteritidis to humans. Chickens serve as natural hosts for S. Enteritidis with the cecum being the principal site of colonization. Cecal colonization of S. Enteritidis in birds leads to the contamination of carcasses during slaughter, contamination of eggshell with feces, and contamination of egg yolk, albumen and shell membranes by transovarian route. Therefore, reducing S. Enteritidis carriage in chickens is critical for reducing human infections. In this Ph. D. dissertation, the efficacy of three natural molecules, caprylic acid (CA), a medium chain fatty acid, and trans-cinnamaldehyde (TC) and eugenol (EG), two phytophenols, were investigated for reducing cecal colonization and fecal shedding of S. Enteritidis in chickens. In addition, cell culture studies, quantitative real-time PCR and DNA microarray were used to elucidate the molecular mechanisms behind the antimicrobial action of TC and EG. Results revealed that prophylactic and therapeutic supplementation of CA, TC and EG in feed significantly reduced S. Enteritidis populations in the cecum and cloaca of treated chickens, compared to the control birds (P \u3c 0.05). Feeding of CA, TC or EG did not adversely affect the body weight, feed intake, pH, or endogenous cecal bacterial population in treated chickens, compared to the negative controls. Follow up studies revealed that the natural molecules reduced S. Enteritidis motility and invasion of avian intestinal epithelial cells, in vitro (P \u3c 0.05). Real-Time quantitative PCR results indicated that the expression of major virulence genes such as hilA, hilD, invF, flhC , and motA in S. Enteritidis was reduced significantly by CA, TC and EG (P \u3c 0.05). The DNA microarray data revealed that the TC and EG exerted antimicrobial effect on S. Enteritidis by multiple mechanisms, including down-regulation of genes responsible for flagellar motility, Salmonella Pathogenicity Island 1, type three secretion system, translocation and effector proteins, hyb virulence operon, outer membrane proteins, cell division, transcription, translation, metabolism, biosynthetic pathways, and terminal electron acceptors. The efficacy of CA, TC and EG in reducing S. Enteritidis in chickens could be utilized in the control of the pathogen in chickens as a pre-harvest treatment for potentially decreasing foodborne diseases caused by the pathogen.

Food Grade Pimenta Leaf Essential Oil Reduces the Attachment of Salmonella enterica Heidelberg (2011 Ground Turkey Outbreak Isolate) on to Turkey Skin

Salmonella attached to the poultry skin is a major source of carcass contamination during processing. Once attached to the poultry skin, it is difficult to detach and inactivate Salmonella by commonly used antimicrobial agents since the pathogen is entrapped deeply in the feather follicles and the crevices on the skin. Essential oils could be natural, safe, and effective alternatives to synthetic antimicrobial agents during commercial and organic processing setup. The present study evaluated the efficacy of pimenta (Pimenta officinalis Lindl.) leaf essential oil (PEO), and its nanoemulsion in reducing Salmonella Heidelberg attachment on to turkey (Meleagris gallopavo) skin during simulated scalding (65°C) and chilling (4°C) steps in poultry processing. A multidrug resistant S. Heidelberg isolate from the 2011 ground turkey outbreak in the United States was used in the study. Results showed that PEO and the nanoemulsion resulted in significant reduction of S. Heidelberg attachment on turkey skin. Turkey skin samples treated with 1.0% PEO for 5 min resulted in >2 log10 CFU/sq. inch reduction of S. Heidelberg at 65 and 4°C, respectively (n = 6; P < 0.05). Similarly, skin samples treated with 1.0% pimenta nanoemulsion (PNE) for 5 min resulted in 1.5- and 1.8- log10 CFU/sq. inch reduction of S. Heidelberg at 65 and 4°C, respectively (n = 6; P < 0.05). In addition, PEO and PNE were effective in reducing S. Heidelberg on skin during short-term storage at 4 and 10°C (temperature abuse) (n = 6; P < 0.05). No Salmonella was detected in the dipping solution containing 0.5 or 1.0% PEO or PNE, whereas a substantial population of the pathogen survived in the control dipping solution. The results were validated using scanning electron -, and confocal - microscopy techniques. PEO or PNE could be utilized as an effective antimicrobial agent to reduce S. Heidelberg attachment to turkey skin during poultry processing

Characterizing the Antimicrobial Function of a Dairy-Originated Probiotic, Propionibacterium freudenreichii, Against Multidrug-Resistant Salmonella enterica Serovar Heidelberg in Turkey Poults

Antimicrobial potential of a dairy-origin probiotic bacteria, Propionibacterium freudenreichii, against multidrug-resistant Salmonella Heidelberg (SH) in turkey poults was determined in the current study. Employing in vitro experiments, two strains (subsp.) of P. freudenreichii: P. freudenreichii freudenreichii B3523 (PF) and P. freudenreichii shermanii B4327 (PS) were tested for their ability to resist low pH (2.5) and bile salts (0.3%). In addition, the ability of the strains to adhere to and invade avian epithelial cells was determined after exposure to Propionibacterium strains followed by SH challenge. Moreover, the antibacterial activity of the strains’ cell-free culture supernatants (CFCSs) were tested against three major foodborne pathogens, including SH. Furthermore, the susceptibility of the strains to common antibiotics used for human therapy was determined. The hemolytic properties of the strains were determined in comparison to Streptococcus pyogenes, a known hemolysis-causing pathogen. Appropriate controls were kept in all studies. Using two in vivo experiments, PF was tested against SH colonization of poult ceca and dissemination to liver and spleen. The four treatment groups were: negative control, PF control (PFC), SH control (SC), and a test group (PFS; PF + SH). The poults in the PFC and PFS groups were inoculated with 1010 CFU ml−1 PF on day 1 through crop gavage and subsequently supplemented through drinking water. On day 7, SC and PFS groups were challenged with SH at 106 CFU ml−1, and after 7 days, cecum, liver, and spleen were collected for determining surviving SH populations. Results indicated that both PF and PS resisted pH = 2.5 and 0.3% bile salts with surviving populations comparable to the control and adhered well onto the avian epithelial cell lines. The strains were susceptible to antibiotics and did not invade the epithelial cells or exhibit hemolytic properties. The CFCSs were highly bactericidal against all tested pathogens. In turkey poults, PF significantly reduced cecal colonization of SH and the dissemination of the pathogen to the liver, compared to the SH challenge controls (P < 0.05). Results revealed that PF, a non-host gastrointestinal tract-derived probiotic, could be an antibiotic alternative to prevent the early colonization of SH in poults, improving the preharvest safety of turkeys

Combating Pathogenic Microorganisms Using Plant-Derived Antimicrobials: A Minireview of the Mechanistic Basis

The emergence of antibiotic resistance in pathogenic bacteria has led to renewed interest in exploring the potential of plantderived antimicrobials (PDAs) as an alternative therapeutic strategy to combat microbial infections. Historically, plant extracts have been used as a safe, effective, and natural remedy for ailments and diseases in traditional medicine. Extensive research in the last two decades has identified a plethora of PDAs with a wide spectrum of activity against a variety of fungal and bacterial pathogens causing infections in humans and animals. Active components of many plant extracts have been characterized and are commercially available; however, research delineating the mechanistic basis of their antimicrobial action is scanty. This review highlights the potential of various plant-derived compounds to control pathogenic bacteria, especially the diverse effects exerted by plant compounds on various virulence factors that are critical for pathogenicity inside the host. In addition, the potential effect of PDAs on gut microbiota is discussed

Combating Pathogenic Microorganisms Using Plant-Derived Antimicrobials: A Minireview of the Mechanistic Basis

The emergence of antibiotic resistance in pathogenic bacteria has led to renewed interest in exploring the potential of plant-derived antimicrobials (PDAs) as an alternative therapeutic strategy to combat microbial infections. Historically, plant extracts have been used as a safe, effective, and natural remedy for ailments and diseases in traditional medicine. Extensive research in the last two decades has identified a plethora of PDAs with a wide spectrum of activity against a variety of fungal and bacterial pathogens causing infections in humans and animals. Active components of many plant extracts have been characterized and are commercially available; however, research delineating the mechanistic basis of their antimicrobial action is scanty. This review highlights the potential of various plant-derived compounds to control pathogenic bacteria, especially the diverse effects exerted by plant compounds on various virulence factors that are critical for pathogenicity inside the host. In addition, the potential effect of PDAs on gut microbiota is discussed

Antibiotic-Resistant Salmonella in the Food Supply and the Potential Role of Antibiotic Alternatives for Control

Salmonella enterica is one of the most ubiquitous enteropathogenic bacterial species on earth, and comprises more than 2500 serovars. Widely known for causing non-typhoidal foodborne infections (95%), and enteric (typhoid) fever in humans, Salmonella colonizes almost all warm- and cold-blooded animals, in addition to its extra-animal environmental strongholds. The last few decades have witnessed the emergence of highly virulent and antibiotic-resistant Salmonella, causing greater morbidity and mortality in humans. The emergence of several Salmonella serotypes resistant to multiple antibiotics in food animals underscores a significant food safety hazard. In this review, we discuss the various antibiotic-resistant Salmonella serotypes in food animals and the food supply, factors that contributed to their emergence, their antibiotic resistance mechanisms, the public health implications of their spread through the food supply, and the potential antibiotic alternatives for controlling them

Combating Pathogenic Microorganisms Using Plant-Derived Antimicrobials: A Minireview of the Mechanistic Basis

The emergence of antibiotic resistance in pathogenic bacteria has led to renewed interest in exploring the potential of plant-derived antimicrobials (PDAs) as an alternative therapeutic strategy to combat microbial infections. Historically, plant extracts have been used as a safe, effective, and natural remedy for ailments and diseases in traditional medicine. Extensive research in the last two decades has identified a plethora of PDAs with a wide spectrum of activity against a variety of fungal and bacterial pathogens causing infections in humans and animals. Active components of many plant extracts have been characterized and are commercially available; however, research delineating the mechanistic basis of their antimicrobial action is scanty. This review highlights the potential of various plant-derived compounds to control pathogenic bacteria, especially the diverse effects exerted by plant compounds on various virulence factors that are critical for pathogenicity inside the host. In addition, the potential effect of PDAs on gut microbiota is discussed

Effect of Chlorine Exposure on the Survival and Antibiotic Gene Expression of Multidrug Resistant Acinetobacter baumannii in Water

Acinetobacter baumannii is a multidrug resistant pathogen capable of causing a wide spectrum of clinical conditions in humans. Acinetobacter spp. is ubiquitously found in different water sources. Chlorine being the most commonly used disinfectant in water, the study investigated the effect of chlorine on the survival of A. baumannii in water and transcription of genes conferring antibiotic resistance. Eight clinical isolates of A. baumannii, including a fatal meningitis isolate (ATCC 17978) (~108 CFU/mL) were separately exposed to free chlorine concentrations (0.2, 1, 2, 3 and 4 ppm) with a contact time of 30, 60, 90 and 120 second. The surviving pathogen counts at each specified contact time were determined using broth dilution assay. In addition, real-time quantitative PCR (RT-qPCR) analysis of the antibiotic resistance genes (efflux pump genes and those encoding resistance to specific antibiotics) of three selected A. baumannii strains following exposure to chlorine was performed. Results revealed that all eight A. baumannii isolates survived the tested chlorine levels during all exposure times (p > 0.05). Additionally, there was an up-regulation of all or some of the antibiotic resistance genes in A. baumannii, indicating a chlorine-associated induction of antibiotic resistance in the pathogen