The occurrence and characteristics of Listeria monocytogenes in commercial and native chicken breeds

The occurrence and characteristics of Listeria monocytogenes in commercial and native chicken breeds

Azza S. El-Demerdash*, Ahmed A. Matter^**, Mona S. Ibrahim^***,

Adel A.A.M. El-Gmaal^*, Salma Salah El-Deen Mohamed^*, Rehab E. Mowafy^****, Amera F. Ebrahem^**

^* Department of Microbiology, Agriculture Research Centre (ARC), Animal Health Research Institute (AHRI), Zagazig Branch, Egypt

^** Department of Poultry Diseases, Agriculture Research Centre (ARC), Animal Health Research Institute (AHRI), Gamasa Branch, Egypt

^*** Department of Poultry Diseases, Agriculture Research Centre (ARC), Animal Health Research Institute (AHRI), Mansoura Branch, Egypt

^**** Department of Pathology, Agriculture Research Centre (ARC), Animal Health Research Institute (AHRI), Zagazig Branch, Egypt

ABSTRACT

The present study was designed to evaluate the potential presence and features of Listeria monocytogenes in different commercial and native poultry breeds. Overall, the isolation of L. monocytogenes strains from different chicken species was 42/550 (7.6%). L. monocytogenes showed high resistance to streptomycin (83.3%) and amoxicillin/clavulanic acid (78.5%) and was highly sensitive to vancomycin, gentamycin, and chloramphenicol. Multiple drug resistance (MAR) index values for 59.5% of the isolates were greater than 0.2. L. monocytogenes strains were screened for five virulence factors associated with genes, namely inlA, actA, hlyA, iap, and inlB. The presence of the five virulence genes were 83.3%, 66.6%, 59.5%, 57.1%, and 45.2% for actA, iap, inlA, hlyA, and inlB, respectively, in the identified L. monocytogenes isolates. Moreover, histopathological examination revealed marked changes in the brain and heart as the most affected tissues. Both the cerebral hemispheres and cerebellum were affected and exhibited marked encephalitis represented by diffuse congestion of cerebral blood vessels. These findings suggest that poultry products may play a role in the zoonotic spread and transmission of multidrug-resistant and virulent L. monocytogenes, which can pose a health risk at human-poultry interface, especially in the absence of stringent hygienic standards and preventive measures.

INTRODUCTION

Listeria monocytogenes (L. monocytogenes), a Gram-positive facultative anaerobic bacillus can cause severe diseases in human and poultry. In chickens, listeriosis has been associated with two major forms of disease: septicemic and encephalitic. The World Health Organization has identified L. monocytogenes as one of four foodborne pathogens causing invasive infections in both humans and animals with a death rate of 20–30%. It can lead to severe listeriosis, which can cause meningoencephalitis, sepsis, fetal infection, or miscarriage in pregnant women (Radoshevich and Cossart, 2018). Consumption of processed chicken and its products, poultry, and poultry products may significantly contribute to the spread and transmission of virulent and multiple drug resistance (MDR) L. monocytogenes to humans (White‏ et al. 2002).  L. monocytogenes has been found at all stages of poultry production and processing (Rothrock et al. 2017). The intestinal colonization of chickens and the presence of L. monocytogenes in their feces is a possible source of the organism, leading to contamination of the environment and litter in the chicken production facility (Dhama et al. 2013). Ingestion, inhalation, or wound contamination are the three main modes of transmission. Symptoms of infection can include sudden death, ataxia, lateral recumbency with leg paddling, twisting and backward neck retraction, and paralysis (Abdel Aziz and Mohamed, 2020).

The overuse of medications in clinical settings and their extensive use as growth promoters for farm animals, as well as increased global trade and travel, which favor the spread of antimicrobial resistance between nations and continents, are all accelerating factors of antimicrobial resistance in L. monocytogenes (Moreno et al. 2014). Among high-risk groups, the multi drug resistant (MDR) L. monocytogenes found in ready-to-eat foods is being regarded as a public health indicator, thus increasing understanding of the laws governing food safety and of medications used in both people and animals is strongly advised (Garedew et al. 2015).

Several virulence factors, which are responsible for microbial invasion and adhesion as well as entry and replication within host cells, have been found to influence the pathogenicity of L. monocytogenes (Matereke and Okoh, 2020). In particular, the inlA and inlB genes promote bacterial uptake by host cells., and the secreted pore-forming toxin listeriolysin O (LLO), disrupts the phagosome to allow bacterial proliferation in the cytosol. It has also been demonstrated that L. monocytogenes internalization into epithelial cells is facilitated by LLO perforating the plasma membrane (Phelps et al. 2018).  In real-time PCR-based studies, the hemolysin gene (hlyA) has been identified as a useful genetic marker for L. monocytogenes (Salim and Othman, 2017). The actin gamma gene (actA), a critical factor in L. monocytogenes persistence in the host and transmission, is a primary virulence determinant (Travier et al. 2013). Finally, the extracellular invasion-associated protein (IAP), encoded by the iap gene, plays a crucial role in the bacterium's virulence and pathogenicity (Soni et al. 2014).

Given its potential to cause a variety of health issues in humans, it is important to understand the prevalence of this bacterium in different breeds. Therefore, this study was designed to study the pathogenicity pattern and antibiotic resistance profiles of L. monocytogenes isolated from various chicken commercial and native breeds in Egypt.

MATERIALS AND METHODS

 Samples collection

Five hundred and fifty randomly collected cloacal swab samples were collected from commercial and native chicken breeds at ages ranging from 8 to 12 weeks. Three hundred samples were isolated from broilers, 150 from layers, and 100 from Baladi chicken farms in Sharkia and Dakahlia Governorates in Egypt during the period from January to June 2023. The samples were collected in sterile plastic bags, kept in ice boxes, and with a minimum delay were transferred to the laboratory to study the presence of Listeria species.

Pathological examinations 

The investigations were performed on samples taken from the brains and hearts of infected birds that had been either slaughtered or found freshly dead. Following a fixation step in 10% buffered neutral formalin, the specimens were cut into paraffin slices 2-3 microns thick and stained with hematoxylin and eosin.  These sections were then inspected under a microscope (Suvarna et al. 2013).

Bacteriological examination

Primary Enrichment: Twenty-five grams of the sample were inoculated into 225 ml of tryptone soya broth (TBS) and incubated aerobically at 30 ± 1C for 24 ± 2 hours. Secondary Enrichment: 0.1 ml of the incubated broth was inoculated into 10 ml of Fraser broth and incubated at 37 ᵒC for 24±2 hours. A loopful from the incubated Fraser broth was streaked onto the following media: ALOA agar; PALCAM agar and Oxford agar plates then the plates were incubated at 37 ᵒC for 48 hours and examined after 24 ± 3 hours. The listeria-like colonies were picked and streaked onto Tryptic Soy agar (TSA) and then, incubated at 37ᵒC for 24 hours. The isolates were morphologically identified by Gram stain and biochemical tests according to ISO 11290-1, (2017).

Antibiogram Profile

The isolated L. monocytogenes strains were subjected to a sensitivity test, using the Kirby-Bauer disk diffusion method. The 11 antibiotic discs belonging to 8 different antibiotic classes included: erythromycin(E), gentamycin (CN), danofloxacin (DA), ampicillin (AMP), sulfamethoxazole/trimethoprim (SXT), chloramphenicol (C), streptomycin (S), doxycycline (DO), amoxycillin/Clavulanic Acid (AMC), vancomycin (VA), and norfloxacin (NOR). The result was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines of 2022.  L. monocytogenes ATCC 7644 was used as a control. The MARI values were calculated according to Krumperman (1983) using the mathematical formula: MARI= a/b (where a is the sum of the test antibiotics the isolates displayed resistance to and b is the total sum of antimicrobial agents used).

Molecular confirmation and genotypic characterization of L. monocytogenes isolates.

 The bacteriologically identified L. monocytogenes isolates were confirmed by PCR using the 16S rRNA gene. Five sets of primers were used for genotypic detection of L. monocytogenes virulence genes: inlA; inlB; hlyA; iap and actA. This was applied on 41 isolated L. m following the QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany), Taq PCR Master Mix Kit and Agarose 1.5%. The specific sequence and amplification are shown in Table 1. Temperature and time conditions of the primers during PCR were as follows initial denaturation at 95 °C for 3 min; 35 cycles each consisting of denaturation at 94 °C for 30 s, annealing at 53 °C for 15 s, and extension at 72 °C for 90 s; and final extension at 72 °C for 7 min.