Antibacterial and anti-biofilm activities of probiotic Lactobacillus plantarum against Listeria monocytogenes isolated from milk, chicken and pregnant women.

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that poses significant risks to public health and food safety. The present study aimed to identify the presence of Listeria spp. in various samples, including pasteurized milk, chicken fillets, and stool samples from pregnant women in Sharkia Governorate, Egypt. Additionally, the study identified the serotypes, virulence-associated genes, antimicrobial resistance patterns, and biofilm formation in L. monocytogenes isolates. Moreover, the antibacterial and anti-biofilm activity of Lactobacillus plantarum ATCC 14917 (L. plantarum) against L. monocytogenes isolates was investigated. A cross-sectional study was conducted from August 2021 to January 2022 to collect 300 samples of pasteurized milk, chicken fillets, and stool from pregnant women admitted to outpatient clinics of hospitals. The results showed that 32.7% of the samples were positive for Listeria spp., including L. innocua (48.9%), L. monocytogenes (26.5%), L. ivanovii (14.3%), L. grayi (5.1%), and L. welshimeri (5.1%). Among all L. monocytogenes isolates, hlyA, actA, inlC, and inlJ virulence-associated genes were detected. However, the virulence genes plcB, iap, and inlA were found in 10 (38.5%), 8 (30.8%), and 25 (96.2%) isolates, respectively. The L. monocytogenes isolates classified into four serotypes (1/2a, 1/2b, 1/2c, and 4b), with 1/2a and 4b each identified in 30.8% of the isolates, while 1/2b and 1/2c were identified in 19.2% of the isolates. All L. monocytogenes isolates showed 100% resistance to streptomycin, kanamycin, and nalidix acid, and 92.3% of isolates showed gentamicin resistance. However, all isolates were susceptible to ampicillin and ampicillin/sulbactam. Multidrug resistance (MDR) was observed in 20 (76.9%) L. monocytogenes isolates. The biofilm formation ability of 26 L. monocytogenes isolates was evaluated at different incubation temperatures. At 4°C, 25°C, and 37°C, 53.8, 69.2, and 80.8% of the isolates, respectively, were biofilm producers. Furthermore, 23.1% were strong biofilm producers at both 4°C and 25°C, while 34.6% were strong biofilm formers at 37°C. Treating L. monocytogenes isolates with L. plantarum cell-free supernatant (CFS) reduced the number of biofilm-producing isolates to 15.4, 42.3, and 53.8% at 4°C, 25°C, and 37°C, respectively. L. plantarum's CFS antibacterial activity was tested against six virulent, MDR, and biofilm-forming L. monocytogenes isolates. At a concentration of 5 µg/mL of L. plantarum CFS, none of the L. monocytogenes isolates exhibited an inhibition zone. However, an inhibition zone was observed against L. monocytogenes strains isolated from pasteurized milk and pregnant women's stools when using a concentration of 10 µg/mL. Transmission electron microscopy (TEM) revealed that L. plantarum CFS induced morphological and intracellular structural changes in L. monocytogenes. In conclusion, this study identified virulent MDR L. monocytogenes isolates with strong biofilm-forming abilities in food products in Egypt, posing significant risks to food safety. Monitoring the prevalence and antimicrobial resistance profile of L. monocytogenes in dairy and meat products is crucial to enhance their safety. Although L. plantarum CFS showed potential antibacterial and anti-biofilm effects against L. monocytogenes isolates, further research is needed to explore its full probiotic potential.

Therapeutic Switching of Rafoxanide: a New Approach To Fighting Drug-Resistant Bacteria and Fungi.

Control and management of life-threatening bacterial and fungal infections are a global health challenge. Despite advances in antimicrobial therapies, treatment failures for resistant bacterial and fungal infections continue to increase. We aimed to repurpose the anthelmintic drug rafoxanide for use with existing therapeutic drugs to increase the possibility of better managing infection and decrease treatment failures. For this purpose, we evaluated the antibacterial and antifungal potential of rafoxanide. Notably, 70% (70/100) of bacterial isolates showed multidrug resistance (MDR) patterns, with higher prevalence among human isolates (73.5% [50/68]) than animal ones (62.5% [20/32]). Moreover, 22 fungal isolates (88%) were MDR and were more prevalent among animal (88.9%) than human (87.5%) sources. We observed alarming MDR patterns among bacterial isolates, i.e., Klebsiella pneumoniae (75% [30/40; 8 animal and 22 human]) and Escherichia coli (66% [40/60; 12 animal and 28 human]), and fungal isolates, i.e., Candida albicans (86.7% [13/15; 4 animal and 9 human]) and Aspergillus fumigatus (90% [9/10; 4 animal and 5 human]), that were resistant to at least one agent in three or more different antimicrobial classes. Rafoxanide had antibacterial and antifungal activities, with minimal inhibitory concentration (MICs) ranging from 2 to 128 µg/mL. Rafoxanide at sub-MICs downregulated the mRNA expression of resistance genes, including E. coli and K. pneumoniae blaCTX-M-1, blaTEM-1, blaSHV, MOX, and DHA, C. albicans ERG11, and A. fumigatus cyp51A. We noted the improvement in the activity of ß-lactam and antifungal drugs upon combination with rafoxanide. This was apparent in the reduction in the MICs of cefotaxime and fluconazole when these drugs were combined with sub-MIC levels of rafoxanide. There was obvious synergism between rafoxanide and cefotaxime against all E. coli and K. pneumoniae isolates (fractional inhibitory concentration index [FICI] values ≤ 0.5). Accordingly, there was a shift in the patterns of resistance of 16.7% of E. coli and 22.5% of K. pneumoniae isolates to cefotaxime and those of 63.2% of C. albicans and A. fumigatus isolates to fluconazole when the isolates were treated with sub-MICs of rafoxanide. These results were confirmed by in silico and mouse protection assays. Based on the in silico study, one possible explanation for how rafoxanide reduced bacterial resistance is through its inhibitory effects on bacterial and fungal histidine kinase enzymes. In short, rafoxanide exhibited promising results in overcoming bacterial and fungal drug resistance. IMPORTANCE The drug repurposing strategy is an alternative approach to reducing drug development timelines with low cost, especially during outbreaks of disease caused by drug-resistant pathogens. Rafoxanide can disrupt the abilities of bacterial and fungal cells to adapt to stress conditions. The coadministration of antibiotics with rafoxanide can prevent the failure of treatment of both resistant bacteria and fungi, as the resistant pathogens could be made sensitive upon treatment with rafoxanide. From our findings, we anticipate that pharmaceutical companies will be able to utilize new combinations against resistant pathogens.

Tamarindus indica Extract as a Promising Antimicrobial and Antivirulence Therapy.

The worldwide crises from multi-drug-resistant (MDR) bacterial infections are pushing us to search for new alternative therapies. The renewed interest in medicinal plants has gained the attention of our research group. Tamarindus indica L. (T. indica) is one of the traditional medicines used for a wide range of diseases. Therefore, we evaluated the antimicrobial activities of ethanolic extract of T. indica. The inhibitions zones, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and fractional inhibitor concentration indices (FICI) against Gram+ve and -ve pathogens were detected. The bioactive compounds from T. indica extract were identified by mass spectroscopy, thin-layer chromatography, and bio-autographic assay. We performed scanning electron microscopy (SEM) and molecular docking studies to confirm possible mechanisms of actions and antivirulence activities, respectively. We found more promising antimicrobial activities against MDR pathogens with MIC and MBC values for Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), i.e., (0.78, 3.12 mg/mL) and (1.56, 3.12 mg/mL), respectively. The antimicrobial activities of this extract were attributed to its capability to impair cell membrane permeability, inducing bacterial cell lysis, which was confirmed by the morphological changes observed under SEM. The synergistic interactions between this extract and commonly used antibiotics were confirmed (FICI values < 0.5). The bioactive compounds of this extract were bis (2-ethylhexyl)phthalate, phenol, 2,4-bis(1,1-dimethylethyl), 1,2-benzenedicarboxylic acid, and bis(8-methylnonyl) ester. Additionally, this extract showed antivirulence activities, especially against the S. aureus protease and P. aeruginosa elastase. In conclusion, we hope that pharmaceutical companies can utilize our findings to produce a new formulation of T. indica ethanolic extract with other antibiotics.

New Insights into Listeria monocytogenes Antimicrobial Resistance, Virulence Attributes and Their Prospective Correlation.

Listeriosis is one of the most common foodborne diseases caused by Listeria monocytogenes (L. monocytogenes). A poor prognosis has been recorded for the invasive listeriosis, especially neurolisteriosis. In several countries throughout the world, foodborne infections with L. monocytogenes exceeded the legal safety limits in animal sourced foods. Therefore, we decided to investigate the variability, virulence and antimicrobial resistance profiles of this pathogen. Both phenotypic and genotypic methods were used for identifying L. monocytogenes isolates and confirming their virulence profiles. The antimicrobial resistances and their correlation analysis with the existence of virulence genes were detected. Additionally, sequencing and phylogenetic analysis based on L. monocytogenes inlA and inlB genes were undertaken. The prevalence rate (11.9%) and the resistance profiles of L. monocytogenes were shocking. The multi-drug resistance (MDR) phenotypes were common among our isolates (64.9%). Fortunately, the resistance phenotypes were always associated with low virulence arrays and the MDR strains possessed low virulence fitness. Herein, the high genotypic and phenotypic diversity of L. monocytogenes isolates and their weak clonality and adaptability highlighted the difficulty in controlling and managing this pathogen. Therefore, it is important to add more restriction guidelines from national authorities on the consumption of ready to eat foods.

Alternative and Complementary Therapies against Foodborne Salmonella Infections.

The limitations in the therapeutic options for foodborne pathogens lead to treatments failure, especially for multidrug-resistant (MDR) Salmonella sp., worldwide. Therefore, we aimed to find alternative and complementary therapies against these resistant foodborne pathogens. Out of 100 meat products samples, the prevalence rate of salmonella was 6%, serotyped only as S. Typhimurium and S. Enteritidis. According to the antibiotic susceptibility assays, the majority of our isolates were MDR and susceptible to cefotaxime. Out of the 13 tested plant extracts, five only showed an inhibition zone in the range of 8-50 mm against both serotypes. Based on their promising activity, the oily extract of cinnamon and aqueous extract of paprika represented the highest potency. Surprisingly, a significant synergistic effect was detected between cinnamon oil and cefotaxime. Depending on Gas Chromatography/Mass Spectrometry (GC-MS), the antimicrobial activity of cinnamon oil was attributed to four components including linalool, camphor, (Z)-3-Phenylacrylaldehyde and its stereoisomer 2-Propenal-3-phenyl. The anti-virulence activities of these compounds were confirmed on the basis of computational molecular docking studies. Accordingly, we recommended the use of cinnamon oil as a food additive to fight the resistant foodborne pathogens. Additionally, we confirmed its therapeutic uses, especially when co-administrated with other antimicrobial agents.