الفهرس 
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Abstract
Listeria monocytogenes is an important small, facultatively anaerobic, non-sporulating, Gram-positive bacteria infecting large varieties of animal species specially ruminants and humans causing Listeriosis. Therefore, this study was conducted to investigate the genotypic characters and molecular differences and identity between Listeria monocytogenes from 3 different forms of infection in sheep.
A total of 290 samples were collected from different sheep cases suspected of being infected with listeriosis in Alexandria (Borg El Arab city) and Marsa Matrouh governorates during the period from May (2017) until June (2019) consisting of; 50 samples from animals have nervous manifestation, 90 samples from animals have abortive signs and 150 samples from animals have septicemic signs. Samples were analyzed by bacteriological methods then confirmed by MALDI-TOF MS, molecular and genetic differentiation and relatedness were estimated by PCR and sequencing methods and finally, antimicrobial resistance profiling and biofilm formation ability were detected.
Out of 290 samples; 15 (5.2%) L. monocytogenes isolates were detected; represented as 8 isolates (2.8%) from nervous form, 4 isolates (1.4%) from abortive and 3 isolates (1.0 %) from septicemic form. The characteristic morphologic appearance of L. monocytogenes isolates on different laboratory media showed that on ALOA agar it gave green-blue colonies surrounded by an opaque halo. Whereas; it gave grayish-green colonies with black center and black halo surrounded them on PALCAM agar. Also, On Oxford agar they were grayish colonies having greenish reflection and surrounded with a black halo.
In vitro detection of the Listeria monocytogenes virulence carried out by detection of hemolytic activity, and CAMP test of the 15 isolated L. monocytogenes. The result of the hemolytic activity on 5% sheep blood agar plate revealed that 12 isolates (80%) showed a narrow zone of β hemolysis. Also, 14 isolates (93.3%) were CAMP test positive with a zone of β - hemolysis at the junction of tested isolate and S. aureus strain.
Among the 15 L. monocytogenes isolates, 14 isolates (93.3%) showed the presence of hlyA, prfA, and inlB virulence-associated genes. however, inlA and 16S rRNA genes were detected in all of them. The sequencing results of inlA and inlB genes indicated that there were some variations in the nucleotides sequence between the 3 L. monocytogenes isolates from the 3 different disease forms in sheep, whereas all of them have belonged to the serotype 1/2a – lineage II.
Out of 15 L. monocytogenes isolates; 7 isolates (46.6%) were MDR with MAR index range from (0.25 to 0.8). Resistance to Penicillin, Cefotaxime and Cephalexin were widespread in most of the isolated L. monocytogenes (60%, 53.3% and 46.7%; respectively), followed by Oxytetracycline resistance (33.3%) then Gentamicin and Ampicillin/sulbactam resistance were (26.7% for both). Resistance to Norfloxacin, Tylosin and Amoxicillin/clavulanic acid was less common (20%, 13.3% and 13.3%; respectively). Finally, Chloramphenicol resistance was rare only (6.7%). However, all of the isolates were sensitive to Ciprofloxacin and Trimthoprim/Sulpha-methoxazole.
By detection of biofilm-formation activity, all tested 15 L. monocytogenes isolates were biofilm-forming bacteria but with different degrees. Out of them, 4 isolates (26.7%) were strong biofilm formers, 4 isolates (26.7%) were moderate biofilm formers and the other 7 isolates (46.6%) attribute as weak producer of biofilm.
from the aforementioned results, it can be concluded that sheep represent a potentially important reservoir for L. monocytogenes in Egypt, causing various clinical manifestations. By PCR and sequencing, very slight molecular differences have been found between the isolates from different disease forms and all of them were belonging to serotype 1/2a_II, so more than one type of clinical syndrome can be caused by the same L. monocytogenes strain that may depend on reasons other than molecular reasons such as; animal age, health condition and if the animal was pregnant, etc.
Also, I investigated the prevalence of virulent, MDR and biofilm-forming L. monocytogenes isolated from samples collected from sheep in the Alexandria and Marsa Matrouh province in Egypt, Unfortunately, the isolated L. monocytogenes were resistant to penicillin, which is the most common antibiotic used in listeriosis treatment in Egypt, suggesting that cross-contamination, or eating undercooked sheep meat or milk represent a potential danger to public health. So, particular attention should be paid to the excessive use of antibiotics in the treatment of humans and animals to avoid the rise of new MDR strains of L. monocytogenes.