Isolation and characterization of bacteriophages for combating multidrug-resistant Listeria monocytogenes from dairy cattle farms in conjugation with silver nanoparticles.

BACKGROUND: This study aims to achieve biocontrol of multidrug-resistant Listeria monocytogenes in dairy cattle farms which poses a severe threat to our socio-economic balance and healthcare systems. METHODS: Naturally occurring phages from dairy cattle environments were isolated and characterized, and the antimicrobial effect of isolated L. monocytogenes phages (LMPs) against multidrug-resistant L. monocytogenes strains were assessed alone and in conjugation with silver nanoparticles (AgNPs). RESULTS: Six different phenotypic LMPs (LMP1-LMP6) were isolated from silage (n = 4; one by direct phage isolation and three by enrichment method) and manure (n = 2; both by enrichment method) from dairy cattle farms. The isolated phages were categorized into three different families by transmission electron microscopy (TEM): Siphoviridae (LMP1 and LMP5), Myoviridae (LMP2, LMP4, and LMP6), and Podoviridae (LMP3). The host range of the isolated LMPs was determined by the spot method using 22 multidrug-resistant L. monocytogenes strains. All 22 (100%) strains were susceptible to phage infection; 50% (3 out of 6) of the isolated phages showed narrow host ranges, while the other 50% showed moderate host ranges. We found that LMP3 (the phage with the shortest tail) had the ability to infect the widest range of L. monocytogenes strains. Eclipse and latent periods of LMP3 were 5 and 45 min, respectively. The burst size of LMP3 was 25 PFU per infected cell. LMP3 was stable with wide range of pH and temperature. In addition, time-kill curves of LMP3 alone at MOI of 10, 1 and 0.1, AgNPs alone, and LMP3 in combination with AgNPs against the most phage-resistant L. monocytogenes strain (ERIC A) were constructed. Among the five treatments, AgNPs alone had the lowest inhibition activity compared to LMP3 at a multiplicity of infection (MOI) of 0.1, 1, and 10. LMP3 at MOI of 0.1 in conjugation with AgNPs (10 µg/mL) exhibited complete inhibition activity after just 2 h, and the inhibition activity lasted for 24 h treatment. In contrast, the inhibition activity of AgNPs alone and phages alone, even at MOI of 10, stopped. Therefore, the combination of LMP3 and AgNPs enhanced the antimicrobial action and its stability and reduced the required concentrations of LMP3 and AgNPs, which would minimize the development of future resistance. CONCLUSIONS: The results suggested that the combination of LMP3 and AgNPs could be used as a powerful and ecofriendly antibacterial agent in the dairy cattle farm environment to overcome multidrug-resistant L. monocytogenes.

Epidemiological study on Listeria monocytogenes in Egyptian dairy cattle farms' insights into genetic diversity of multi-antibiotic-resistant strains by ERIC-PCR.

Listeria monocytogenes (L. monocytogenes) is frequently detected in ruminants, especially dairy cattle, and associated with the sporadic and epidemic outbreak of listeriosis in farms. In this epidemiological study, the prevalence, virulence, antibiotic resistance profiles, and genetic diversity of L. monocytogenes in three Egyptian dairy cattle farms were investigated. The risk factors associated with the fecal shedding of L. monocytogenes were analyzed. The L. monocytogenes strains from the three farms were categorized into distinct genotypes based on sampling site and sample type through enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). A total of 1896 samples were collected from animals, environments, and milking equipment in the three farms. Results revealed that 137 (7.23%) of these samples were L. monocytogenes positive. The prevalence of L. monocytogenes in the animal samples was high (32.1%), and the main environmental source of prevalent genotypes in the three farms was silage. For all sample types, L. monocytogenes was more prevalent in farm I than in farms II and III. Risk factor analysis showed seasonal variation in production hygiene. For all sample types, L. monocytogenes was significantly more prevalent in winter than in spring and summer. The level of L. monocytogenes fecal shedding was high likely because of increasing age, number of parities, and milk yield in dairy cattle. Two virulence genes, namely, hlyA & prfA, were also detected in 93 strains, whereas only one of these genes was found in 44 residual strains. Conversely, iap was completely absent in all strains. The strains exhibited phenotypic resistance to most of the tested antibiotics, but none of them was resistant to netilmicin or vancomycin. According to sample type, the strains from the animal samples were extremely resistant to amoxicillin (95.2%, 80/84) and cloxacillin (92.9%, 78/84). By comparison, the strains from the environmental samples were highly resistant to cefotaxime (86.95%, 20/23). Furthermore, 25 multi-antibiotic resistance (MAR) patterns were observed in L. monocytogenes strains. All strains had a MAR index of 0.22-0.78 and harbored antibiotic resistance genes, including extended-spectrum ß-lactamase (blaCTX-M [92.7%] and blaDHA-1 [66.4%]), quinolones (qnrS [91.2%], qnrA [58.4%], parC [58.4%], and qnrB [51%]), macrolides (erm[B] [76.6%], erm(C) [1.5%], and msr(A) [27%]), trimethoprim (dfrD [65.7%]), and tetracyclines (tet(M) [41.6%], tet(S) [8%], and int-Tn [26.3%]). ERIC-PCR confirmed that the strains were genetically diverse and heterogeneous. A total of 137 isolated L. monocytogenes strains were classified into 22 distinct ERIC-PCR groups (A-V). Among them, ERIC E (10.2%) was the most prevalent group. These results indicated that environment and milking equipment served as reservoirs and potential transmission ways of virulent and multidrug-resistant L. monocytogenes to dairy animals, consequently posing threats to public health. Silage is the main environmental source of prevalent genotypes on all three farms. Therefore, hygienic measures at the farm level should be developed and implemented to reduce L. monocytogenes transmission inside dairy cattle farms.

Prevalence of multidrug resistant Salmonella spp. in dairy products with the evaluation of the inhibitory effects of ascorbic acid, pomegranate peel extract, and D-tryptophan against Salmonella growth in cheese.

The present study aimed at investigation of the prevalence and antimicrobial susceptibility of Salmonella spp. in the retailed dairies in Egypt. Besides, the inhibitory effects of some natural additives, including, ascorbic acid, pomegranate peel extract, and D-tryptophan against the isolated Salmonella were evaluated using soft cheese as a food matrix. To reach to this end, different Egyptian retail dairy products were investigated; 30 samples of each product were analyzed. Kariesh cheese samples had the highest Salmonella prevalence rate at 16.67%, followed by market raw milk, and bulk tank milk at 6.66% each, and white soft cheese at 3.33%. Serological examination exhibited 5 different Salmonella serotypes, namely S. Enteritidis, S. Typhimurium, S. Virchow, S. Larochelle, and S. Apeyeme. Antimicrobial susceptibility testing indicated that 100% of the isolates possessed resistance to erythromycin, oxacillin, and nalidixic acid. Some isolates of S. Typhimurium and S. Enteritidis were resistant to all 14 examined antibiotics. Isolates of S. Enteritidis obtained in this study were used to contaminate the freshly prepared soft cheese. Treatment of the artificially Salmonella-contaminated soft cheese with pomegranate peel extract, ascorbic acid, and D-tryptophan revealed a significant (P < 0.05) reduction in Salmonella growth in a dose-dependent manner. Therefore, the examined natural additives can be viewed as a promising new line of preservatives for dairy industry.

Serological Investigation and Epidemiological Analysis of Bovine Leptospirosis in Egypt.

Bovine leptospirosis is a bacterial zoonotic disease of worldwide distribution. Little information is available regarding the occurrence of the disease in the Nile Delta provinces, Egypt. The present study investigated the seroprevalence of leptospirosis among cattle from Dakahlia province, Northern Egypt, and identified the individual variables factors associated with infection. To this end, a total of 600 serum samples from cattle of small stakeholders with various clinical manifestations possibly associated with leptospirosis were collected from different localities across Dakahlia province, Egypt. Sera were examined serologically via ELISA to investigate the occurrence of the disease among animals. Chi-square test and multivariable logistic regression analyses were applied to determine the association between hypothesized risk factors and the disease. Interestingly, our findings showed that 39.33% of the examined sera were positive for Leptospira antibodies, with significant differences among different localities. In addition, statistical analysis showed significant differences among age groups. Notably, the highest prevalence rate (22%) was observed in those aged between 3 and 5 years (p < 0.0001), whereas the lowest prevalence (2.66%) was reported in cattle <1 year old (p < 0.0001). Moreover, females had a significantly higher prevalence rate (35.33%) than males (4%) (p < 0.0001). Furthermore, our results showed significant differences in the occurrence of infection and reported clinical signs (p < 0.0001). Multivariable logistic regression identified repeated breeder and drop milk yield as the best predictors for prediction of ELISA results and linear discriminant analysis (LDA) model showed that overall classification accuracy of ELISA result using clinical signs and demographic data as predictors was 70.7%. The current study concluded a relative high prevalence of leptospirosis among cows bred in movable herds and households in the studied area and that age, repeated breeder and drop milk yield can be considered major risk factors associated with infection.

Immunostimulant effect of a mixed herbal extract on infectious bursal disease virus (IBDV) vaccinated chickens in the context of a co-infection model of avian influenza virus H9N2 and IBDV.

This study was conducted to assess the comparative effects of a mixed herbal extract (MHE) containing Ocimum sanctum, Withania somnifera, Emblica officinalis, Tinospora cordifolia, Mangifera indica, and Asphaltum (shilajit) on infectious bursal disease virus (IBDV)-vaccinated (VAC) chickens infected with IBDV and avian influenza virus (AIV) H9N2. The experiment included three groups (G1-G3): G1, the negative control group; G2, the VAC + challenged (Ch) group; and G3, the VAC + Ch + MHE group. MHE was orally administered continuously for 5 weeks post-vaccination (PV) with IBDV at 12 days of age, and the chicks were simultaneously challenged with virulent IBDV (intraocularly) and AIV H9N2 (intranasally) at 21 days PV. Blood and tissue samples as well as tracheal and cloacal swabs were gathered at different times PV and post-challenge. Immunological and haematological parameters, histopathological lesions, relative organ weights and final live weights revealed significant differences (P ≤ 0.05) between G2 and G3 groups. Furthermore, in the G3 group, the protection rates, ELISA and HI titers and CD4+/CD8+ ratio were significantly increased, whereas viral shedding titers and the heterophil/lymphocyte ratio were decreased. In conclusion, the oral administration of the mixed herbal extract for 5 weeks can stimulate the immune response to IBDV vaccination and relieves the pathogenicity of an AIV H9N2 and IBDV co-infection in chickens.