Diffusion methods, including agar disk-diffusion and agar well-diffusion, as well as dilution methods such as broth and agar dilution, are frequently employed to evaluate the antimicrobial capacity of extracts and essential oils (EOs) derived from Origanum L., Syzygium aromaticum, and Citrus L. The results are reported as inhibition diameters (IDs) and minimum inhibitory concentrations (MICs), respectively. In order to investigate potential sources of variability in antimicrobial susceptibility testing results and to assess whether a correlation exists between ID and MIC measurements, meta-analytical regression models were built using in vitro data obtained through a systematic literature search. The pooled ID models revealed varied bacterial susceptibilities to the extracts and in some cases, the plant species and methodology utilised impacted the measurements obtained (p < 0.05). Lemon and orange extracts were found to be most effective against E. coli (24.4 ± 1.21 and 16.5 ± 0.84 mm, respectively), while oregano extracts exhibited the highest level of effectiveness against B. cereus (22.3 ± 1.73 mm). Clove extracts were observed to be most effective against B. cereus and demonstrated the general trend that the well-diffusion method tends to produce higher ID (20.5 ± 1.36 mm) than the disk-diffusion method (16.3 ± 1.40 mm). Although the plant species had an impact on MIC, there is no evidence to suggest that the methodology employed had an effect on MIC (p > 0.05). The ID–MIC model revealed an inverse correlation (R² = 47.7%) and highlighted the fact that the extract dose highly modulated the relationship (p < 0.0001). The findings of this study encourage the use of extracts and EOs derived from Origanum, Syzygium aromaticum, and Citrus to prevent bacterial growth. Additionally, this study underscores several variables that can impact ID and MIC measurements and expose the correlation between the two types of results.

Biopreservation strategies such as the use of Mediterranean plant extracts to ensure food safety are promising to deal with the emergence of antimicrobial resistances and the overreliance on food chemical additives. In the last few decades, antimicrobial susceptibility testing (AST) for evaluating the in vitro antibacterial potential of plant extracts against the most relevant foodborne pathogens has been widely reported in the literature. The current meta-analysis aimed to summarise and analyse the extensive evidence available in the literature regarding the in vitro antimicrobial capability of Allium, Ocimum and Thymus spp. extracts against foodborne pathogens. A systematic review was carried out to gather data on AST results of these extracts against Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli and Bacillus cereus, including inhibition diameters (ID) and minimum inhibitory concentrations (MIC). A total of 742 records were gathered from a raw collection of 2,065 articles. Weighted mixed-effect linear models were adjusted to data to obtain pooled ID, pooled MIC and the relationship between both model estimations and observations. The pooled results revealed B. cereus as the most susceptible bacteria to Allium sativum (pooled ID = 20.64 ± 0.61 mm) by diffusion methods and S. aureus (pooled MIC = 0.146 mg/mL) by dilution methods. Diffusion methods did not yield conclusive results for Ocimum spp. extracts; however, the lowest pooled MIC was obtained for S. aureus (0.263 mg/mL). Among the foodborne pathogens evaluated, B. cereus showed the highest sensitivity to Thymus spp. extracts by both diffusion and dilution methods (pooled ID = 28.90 ± 2.34 mm and MIC = 0.075 mg/mL). The methodology used for plant extraction was found to not significantly affect MIC values (p > 0.05). Overall, the antimicrobial effectiveness of the studied extracts against Gram-positive and Gram-negative bacteria was demonstrated. Finally, the robustness of the meta-regression model was confirmed, also revealing an inversely proportional correlation between the ID and MIC measurements (p < 0.0001). These results provide a robust scientific basis on the factors affecting the in vitro antimicrobial efficacy of extracts from Mediterranean plants. They also provide valuable information for stakeholders involved in their industrial application in food, including producers, regulatory agencies and consumers which demand green-labelled foods.

Lactic acid bacteria (LAB) are increasingly recognized for their role in food biopreservation due to their ability to synthesize antimicrobial compounds. Milk naturally harbors a wide variety of LAB, offering a promising source for identifying strains with biopreservative potential. This study investigated the antagonistic effects, safety characteristics, and technological properties of LAB strains isolated from traditionally fermented milk. Thirty-two dairy samples were analyzed, and the resulting LAB isolates were screened for inhibitory activity against Listeria monocytogenes CECT 4032 and Staphylococcus aureus CECT 976 using agar spot and well diffusion assays. All tested strains exhibited strong antimicrobial effects, with particularly notable inhibition of L. monocytogenes. After phenotypic screening, five representative isolates were selected for molecular identification and further assessment of safety-related attributes, functional capabilities, auto- and co-aggregation properties. 16S rRNA gene sequencing revealed that four strains belonged to the genus Enterococcus, specifically, one E. faecium and three E. durans, while one was classified as a Lactococcus species. Moreover, none of the strains showed proteolytic or lipolytic activities which highlights their potential use in dairy fermentation processes.