Microbiological Research 258 (2022) 126980 Contents lists available at ScienceDirect Microbiological Research journal homepage: www.elsevier.com/locate/micres Antibacterial efficacies and time-kill kinetics of indigenous Ghanaian spice extracts against Listeria monocytogenes and some other food-borne pathogenic bacteria Christian Xedzro a,*,1, Kwaku Tano-Debrah b, Hiroyuki Nakano a a Laboratory of Food Microbiology and Hygiene, Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739- 8528, Japan b Department of Nutrition and Food Science, School of Biological Science, University of Ghana, P.O. Box LG 134, Accra, Ghana A R T I C L E I N F O A B S T R A C T Keywords: Increase in food-borne outbreaks has become public health concern worldwide. Exploitation of the antimicrobial Dietary spices properties of dietary spices has become important pharmaceutical tool for controlling food-borne pathogens. This Antibacterial activity study aimed at evaluating the antibacterial potentials of Ghanaian spices against Listeria monocytogenes and other Time-kill kinetics prevalent food-borne pathogens. In preliminary studies, Listeria was isolated from some food samples. The overall L. monocytogenes GC-MS prevalence of Listeria spp. was 23% (13/56). Of the 56 samples examined, 7% showed pathogenic potential for Ghana L. monocytogenes. Different solvent extracts of thirteen spices namely Calabash nutmeg, West African black pepper, Aidan, Grains of paradise, Negro pepper, Aniseed, African locust bean, Cinnamon, Black pepper, Clove, Cayenne, Basil, and Rauvolfia were tested for their potentials to inhibit clinical and isolated strains of L. monocytogenes using qualitative and quantitative antimicrobial assay methods. Only clove and negro pepper among the thirteen different solvent extracts showed bacteriostatic and bactericidal activity against L. monocytogenes indicated by minimum inhibitory concentrations ranging from 0.05% to 0.4% and minimum bactericidal concentrations ranging from 0.1% to > 0.4% under experimental conditions. Time-kill study demonstrated listericidal activity of ethanolic clove and negro pepper extracts indicated by absolute mortality of more than 3 log units at 2x MIC and 4x MIC. GC-MS analysis revealed three and eight major chemical compo- nents present in clove and negro pepper respectively. Staphylococcus aureus, methicillin-resistant S. aureus, Ba- cillus cereus, and B. subtilis also showed satisfactory susceptibilities to ethanolic extracts with MIC ranging from 0.025% to > 0.8%. In general, negro pepper showed broad activity eliciting inhibitory effects against all the tested pathogens. The findings suggest that clove and negro pepper may be promising antibacterial candidates for the decontamination and control bacterial pathogens in food and food supply chain. 1. Introduction vegetables (Szymczak et al., 2020). It can grow at chilling temperatures and has biofilm forming ability on both biological and non-biological Listeria monocytogenes is a Gram-positive pathogenic bacterium surfaces. It is tolerant to osmotic stress (Osaili et al., 2011), which implicated in listeriosis, a severe food-borne illness characterized by makes its elimination difficult in the food industry and ultimately septicemia, perinatal infections, meningitis, spontaneous abortion, and causing cross-contamination. Listeria spp. contribute significantly to gastroenteritis (Barre et al., 2016). The illness has high fatality rate global food-borne diseases. Approximately, 48 million people after ranging from 30% to 75%, particularly affecting elderly people, new- consuming contaminated food develop various symptoms of illness, borns, pregnant women, and immunocompromised individuals. The with 128,000 hospitalization and 3,000 deaths annually; of which 1,600 pathogen is mostly transmitted through fecal-oral route (Nayak et al., are listeriosis cases that results in about 260 deaths (Moura et al., 2016). 2015) and has been isolated from various foods, including meat, fish and With increasing number in the cases of listeriosis, health officials are seafood products, raw and pasteurized milk products, salads, fruits, and developing strategies to suppress the growth of Listeria in food. Also, * Corresponding author. E-mail address: ckkxedzro@gmail.com (C. Xedzro). 1 ORCID iD orcid.org/0000-0002-6631-8578 https://doi.org/10.1016/j.micres.2022.126980 Received 29 September 2021; Received in revised form 29 December 2021; Accepted 5 February 2022 Available online 9 February 2022 0944-5013/© 2022 Elsevier GmbH. All rights reserved. C. Xedzro et al. M i c r o b i o l o g i c a l R e s e a r c h 258 (2022) 126980 food-borne pathogenic bacteria such as Escherichia coli, Staphylococcus this study was to investigate antibacterial efficacies of different solvent aureus, Vibrio cholerae among others, are contributing factors to global extracts of 13 Ghanaian spices against isolated and clinical strains of resurgence in the outbreaks of food-borne illnesses. Food industries, L. monocytogenes. The extracts were also tested against S. aureus, with the development of advanced processing techniques, apply chem- methicillin-resistant S. aureus, Bacillus cereus, B. subtilis, which are also ical antibacterial preservatives (Chen et al., 2019) such as sodium prevalent food-borne pathogens of importance. benzoates, nitrates, nitrites, etc., to retard the proliferation of some of these bacterial pathogens in food to increase the shelf life (Wang et al., 2. Materials and methods 2018). However, these preservatives are not widely accepted by con- sumers due to perceived harmful effects on the health of human popu- 2.1. Acquisition and extraction of plant material lace as for instance, the implicated serum glycine decline and carnitine deficiency associated with sodium benzoates (Lennerz et al., 2015), the Thirteen plant materials (spices) locally cultivated in Ghana (Gha- risk of gastric cancer associated with nitrates and nitrites (Song et al., naian origin) were purchased from popular spice markets in March 2015), and other effects that may be related. 2019. Before sample collection, a preliminary survey was conducted Antibiotics emerged at around 1930s and have been used extensively with the spice dealers who had close contact with the farmers in the to control food-borne bacterial infections in clinical practice (Chen et al., various regions to authenticate and ascertain the source of the spices. 2019). Despite the massive effort to control bacterial infections, evolu- Spices were chosen based on their widespread use in local community tionary transformation and variation have led to the emergence of (use for cooking and use in folk medicine). The collected samples were bacterial resistance to know antibiotics (Costa et al., 2017). Molecular disinfected, air dried in a shade and pulverized to increase the surface research has shown that resistance to antibiotics emanated from the area. Each sample was weighed and soaked in different extraction sol- acquisition and dissemination of exogenous genes through genomic vents (99.5% ethanol, 99% methanol, and 99% dimethyl sulfoxide islands and mobile genetic elements (Partridge et al., 2018) which can DMSO; Nacalai tesque, INC, Kyoto, Japan) in the ratio 1:9 w/v. The be integrated into bacterial genomes and resistance cassettes leading to mixtures were subjected to shaking at 25 ◦C for 48 h. For distilled water cumulative drug resistance (Cerezales et al., 2020). The development of extracts, weighed samples were soaked in 80 ◦C distilled water for 30 antibiotic resistance and side effects of some antibiotics and chemical min with intermittent shaking. Filtration was performed using Whatman preservatives are pressing factors triggering the search for alternative filter paper (110 mm; Toyo Roshi Kaisha, Ltd, Japan) and the filtrate antibacterial agents. Novel alternative agents could include the use of subsequently centrifuged at 9500 rpm for 15 min at 4 ◦C. The super- better selectivity and low toxicity antimicrobial peptides. Despite these natants (10% extracts) were obtained and stored at 10 ◦C until use. advantages, short plasma half-life, oral bioavailability, and high cost of production have limited their applications (Liscano et al., 2020; Otvos 2.2. Isolation of Listeria from food and Wade, 2014). Furthermore, bacteriophage endolysins have also emerged as an alternative. However, extra measures to avoid contami- 2.2.1. Sample collection nation, higher cost of production, and the great deal of time needed to Three categories of food sample were considered in this study. A total grow host bacterial strains in high volumes to produce high yield of of 56 food samples, comprising of raw chicken meat (n = 18), some phage are a cumbersome concept (Murray et al., 2021). Focus is now on Ready-to-Eat (RTE) food (sandwish, vegetable salad, coleslaw, maze- natural antimicrobials among plant origin for food preservation. Thus, soba, tuna macaroni salad, grilled sausage, grilled pork, sushi, cooked the investigation of certain plant species for their antimicrobial capac- fish in sauce, grilled fish, and sprout, n = 27), and some dairy products ities as ideal candidates to explore novel antibacterial drugs may yield (cheese, pasteurized milk, yoghurt, and milk powder, n = 11) were useful results even against antibiotic-resistant bacterial strains. randomly sampled from eight retail facilities in Higashi-Hiroshima, Naturally based antibacterial agents such as culinary spices are po- Japan from July 2019 to October of the same year. Three samples tential alternatives. They have been applied to food since ancient times, each of raw chicken and RTE, and two or one dairy product were not only as preservatives or flavoring agents, but also used in folk randomly sourced on different occasions from different sites, and then traditional system of medicine (Shan et al., 2007). Additionally, they are transported on ice packs to the laboratory for microbiological exami- Generally Recognized as Safe (GRAS) for human consumption. They nation. The samples were selected based on commonly known foods possess biologically active compounds acting as antimicrobials, antiox- implicated in the spread of Listeria. idants, and anticarcinogens. The biological and medical effects of these plant origins are mainly due to the intricate products of secondary 2.2.2. Enrichment and isolation performances metabolism. Among these metabolites, terpenoids quinones, phenolics ISO 11290–1:2017 method involving a double enrichment procedure and phenolic acids, lectins, polyacetylenes, alkaloids, tannins, and fla- was employed to isolate Listeria species from the various food categories. vonoids (Nabavi et al., 2015; Witkowska et al., 2013) are known to be Briefly, primary enrichment of 25 g test portion of each sample was done antimicrobial agents which are involved in multiple antimicrobial ac- in 225 mL liquid broth, containing reduced concentration of enrichment tions such as cell wall damage, inhibition of extracellular enzymes (Takó and selective supplements (half-strength Fraser broth) at 30 ◦C for 24 h. et al., 2020), leakage of intracellular components, modification of fatty This was followed by secondary enrichment of 0.1 mL aliquot obtained acids, destruction of protein translocation as well as changes in the from primary enriched culture in 10 mL broth, containing full concen- synthesis of DNA and RNA (Ceruso et al., 2020; Shan et al., 2007; Wit- tration of enrichment and selective supplements (full-strength Fraser kowska et al., 2013) when exposed against microorganisms. broth) at 37 ◦C for 48 h. For liquid samples, 10 mL test portion was In Ghana, spices are used in a variety of ways such as flavorings in employed in 90 mL of half-strength Fraser broth followed by second traditional drinks, seasonings, ingredients for cooking, and in folkloric enrichment. One loop full of each secondary enriched broth cultures was medicine to treat female infertility, hemorrhoids, and epilepsy (Abbiw, surface streaked on two selective agar plates (ALOA and PALCAM) fol- 1990). Recently, there has been a considerable emphasis on studies lowed by incubation at 37 ◦C for 24–48 h. Characteristic blue-green involving spices as means of controlling bacterial pathogens (Kayira and colonies surrounded by opaque halo on ALOA and gray-black colonies Nakano, 2020; Mostafa et al., 2018; Sharma et al., 2017). While there is surrounded by diffuse halo on PALCAM were obtained for purification a growing knowledge on antimicrobial efficacy of spices against on tryptic soy yeast extract agar (TSYEA) for identification tests. food-borne bacterial pathogens, there is limited information on Gha- naian spices against such pathogens. Furthermore, L. monocytogenes as 2.2.3. Phenotypic and biochemical profile an emerging food-borne pathogen in Ghana has not been tested with Various mandatory confirmation tests were performed on isolated some plant materials to any extent before. Accordingly, the purpose of colonies from TSYEA following the standard to ascertain the phenotypic 2 C. Xedzro et al. M i c r o b i o l o g i c a l R e s e a r c h 258 (2022) 126980 and biochemical characteristics of the isolates. The Gram’s staining was was seeded with 5 mL of agar previously inoculated with the bacterial performed followed by microscopic examination and catalase test. culture. Sterilized paper disks (8 mm in diameter; Tokyo Roshi kaisha, Characteristic Gram-positive short rods or coccobacilli which showed Ltd. Japan) impregnated with 50 µL of each extract were placed on the effervescence upon addition of 3% hydrogen peroxide (catalase posi- surface of each agar. The plates were kept in the fridge at 10 ◦C for 2 h to tive) were sub-cultured in non-selective Brain Heart Infusion (BHI) allow diffusion of active agents into the agar then incubated at 35 ◦C for medium at 25 ◦C for 18–24 h. 24 h. Extraction solvents with their respective concentrations served as Motility was performed by spotting a drop of BHI culture on a sterile negative controls. The antibacterial efficacies of the extracts were glass slide followed by examination under a compound microscope. indicated by clear zone of inhibition surrounding the paper disk. Typical Listeria species exhibited tumbling motility and are short rods. Experimental replicates were established for three independent runs and To confirm the results, an alternative test for motility was performed. diameter of inhibition zones computed as means ± standard deviations. Briefly, semi-solid nutrient agar tubes containing 0.4% agar were stab- bed with an inoculating needle and incubated at 25 ◦C for 5 days and 2.3.3. Determination of minimum inhibitory concentrations (MICs) motility observed. Listeria species were motile as observed by umbrella- The agar dilution method was employed to determine MIC as like growth patterns and growth away from the stabbed line. described by Klančnik et al. (2010) with minor modifications. Briefly, Furthermore, the isolates were subjected to Voges-Proskauer and varying concentrations of each spice extract were diluted in 20 mL sugar fermentation tests. For Voges-Proskauer test, 3 mL tubes of VP molten TSA or NA tempered to 52 ◦C to obtain a desired final concen- medium were inoculated and incubated at 37 ◦C for 24 h. After incu- tration of 0.025%, 0.05%, 0.1%, 0.2%, and 0.4%. Ethanolic extracts bation, 3 drops each of 5% α-naphthol and 40% potassium hydroxide were tested up to 0.8% for all other pathogens except L. monocytogenes. solution were added and allowed to stand for 15 min. A positive reaction Bacterial cultures (106 CFU/mL) were streaked on solid TSA was indicated by red band layer on top of the medium. Typical Listeria (L. monocytogenes) or NA (S. aureus, B. cereus, B. subtilis, and MRSA). species are VP-positive. Agar plates were incubated under aerobic condition at 35 ◦C for 24 or Sugar fermentation test was performed in test tubes containing 48 h. The MIC was defined as the lowest concentration of the extracts in bromocresol blue agar base supplemented with 5% each of L-rhamnose, agar that showed no observable growth (i.e., perfect inhibition) after 24 D-xylose, D-glucose, and D-mannitol. The tubes were inoculated with or 48 h. Extraction solvents in amounts corresponding to highest con- TSYEA cultures and incubated at 37 ◦C for 24–28 h. Fermentation of centration (4% or 8%) in extract diluted assay and inoculated agar plates sugar (acid formation) was indicated by change in color from blue to without added spice extracts were used as negative controls. yellow. 2.3.4. Determination of minimum bactericidal concentrations (MBCs) 2.2.4. Pathogenicity confirmation test with hemolytic S. aureus The macro dilution broth method as described by Kayira and Nakano Christie Atkins Munch Peterson (CAMP) test was performed to (2020) was used to determine MBC with some adjustments. A set of test observe the degree of hemolysis on Columbia agar supplemented with tubes holding 10 mL of TSB or NB were diluted with different concen- 5% sheep blood. The agar was streaked with β-hemolytic S. aureus tration of the various solvent extracts to a desired final concentration of (209P) and then with patterns of perpendicular streaks with Listeria 0.025%, 0.05%, 0.1%, 0.2%, and 0.4%. Ethanolic extracts were tested isolates following incubation at 37 ◦C for 24 h. Positive reaction was up to 0.8% for all other pathogens except L. monocytogenes. Each con- indicated by development of β-hemolysis at the intersection of Listeria centration was inoculated with bacterial cultures (106 CFU/mL) and isolates with S. aureus showing synergistic effect of haemolysins for then incubated at 35 ◦C for 24 h. Control experiments were broth test CAMP positive reaction. tubes containing 4% or 8% solvent and those without added plant extract. After incubation, the preparations were sub-cultured by 2.3. Antibacterial assay spreading 0.1 mL aliquot on solid TSA or NA plates and incubated at 35 ◦C for 24 h and examined for bacterial growth. The MBC was defined 2.3.1. Test bacterial strains and growth conditions as the lowest concentration of the extract that demonstrated 99.9% or The test pathogens were clinical Gram-positive isolates obtained 100% reduction of the bacterial growth (Das et al., 2010; Sharma et al., from Hiroshima City Institute of Public Health (HCIPH), Hiroshima, 2012). Japan. They include L. monocytogenes (L. m. AS-1; serotype 4b), L. monocytogenes (L. m. M24–1; serotype 1/2c), B. cereus (IFO 3457), B. 2.3.5. Time-kill kinetics assay subtilis (BF), S. aureus (209P), and methicillin- resistant S. aureus The killing rate and the extent of antibacterial action by ethanolic (MRSA). Four L. monocytogenes strains (Lis C1, Lis C2, Lis C3, and Lis C4) spice extracts against L. monocytogenes was determined by time-kill ki- isolated from raw chicken meat in this study as described above were netics assay method previously described by Bag et al. (2012) with also included. All L. monocytogenes strains were cultivated in tryptic soy minor modifications. Briefly, overnight TSB cultures (24 h) of cocktail of broth or agar (TSB, TSA, Eiken Chemical Co., Ltd, Japan). The assay clinical L. monocytogenes (4b and 1/2c) strains were washed three times medium used for growing the remaining bacteria strains was nutrient by centrifugation at 1000 rpm for 5 min in physiological Phosphate broth or agar (NB, NA, Eiken Chemical Co., Ltd, Japan). A single isolated Buffer Saline (PBS) pH 7.4. The cells were harvested, resuspended in colony from 24 h old TSA or NA plates was suspended in 10 mL of TSB, and exposed to concentrations equal to 0.5x MIC, 1x MIC, 2x MIC, appropriate broth medium and incubated aerobically at 35 ◦C for 24 h and 4x MIC of clove and negro pepper extracts. Samples were taken at 0, under static condition. Prior to antibacterial testing, turbidity of inoc- 2, 4, 6, 8, 10, and 24 h and serially diluted in PBS and spread-plated. The ulum was standardized to match 0.5 McFarland standard. Briefly, 1 mL plates were incubated at 37 ◦C for 24 h. Inoculum without spice extract aliquot broth culture was diluted in sterile TSB or NB medium to final was plated as control. The procedure was repeated in three independent concentration of 106 CFU/mL (OD620 = 0.08–0.1). runs for each extract and the means expressed as log10 CFU/mL. Time-kill curves were generated by plotting log10 CFU/mL against time. 2.3.2. Screening approach for antibacterial activity The decrease in log10 CFU/mL > 3 log units indicate listericidal effect of The various solvent extracts (10%) were used for evaluation of the extracts. antibacterial activity against L. monocytogenes employing Kirby-Bauer disk diffusion method as described by Klančnik et al. (2010) with 2.4. GC-MS analysis of antilisterial extracts minor modifications. Only ethanolic extracts were tested against S. aureus, MRSA, B. cereus, and B. subtilis. Ten mL of molten TSA or NA Ethanolic extracts of clove and negro pepper were analysed for their was poured into a sterile petri dish to form a base layer. The base layer chemical constituents using modified GC-MS analysis method (Yamuna 3 C. Xedzro et al. M i c r o b i o l o g i c a l R e s e a r c h 258 (2022) 126980 et al., 2017) on equipment model JMS-T100GCV "AccuTOF GCv 4G" 3.3. Screening approach for antimicrobial activity (JEOL Ltd., Tokyo, Japan) equipped with HP-5 MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm). For GC-MS All the 13 different solvent spice extracts were screened for their spectroscopic detection, an electron ionization system with ionization antimicrobial activities against L. monocytogenes using disk diffusion energy of EI+ (70 eV, 300 µA) was used. Helium gas (99.999%) was used method. The antimicrobial activities are presented as diameter of inhi- as a carrier gas at a constant flow rate of 1 mL/min. Ion source tem- bition zones (DIZ) in Tables 4 and 5. The results revealed that the ex- perature and injector temperature were set to 250 ◦C and 300 ◦C, tracts have significant (p < 0.05) but variable potency in suppressing respectively. GC oven temperature was programmed from 50 ◦C to microbial growth. Only ethanol and methanol extracts of negro pepper 150 ◦C and then held isothermally for 10 min and finally raised to 300 ◦C among the different extracts demonstrated inhibitory activity against at 10 ◦C/min. 1 µL of diluted sample (1/100, v/v, in ethanol) was L. monocytogenes with DIZ ranging from 9.0 mm to 10.3 mm. Similarly, injected into the column in a split mode with a split ratio 10:1. ethanol, methanol, and DMSO extracts of clove showed significant (p < 0.05) antibacterial efficacy against L. monocytogenes with DIZ ranging 2.4.1. Identification of chemical constituents from 10.8 mm to 13.3 mm. Moreover, distilled water extract of clove The chemical compounds in the extracts were identified based on GC revealed satisfactory activity (9.5–11.2 mm) against the test pathogens retention times on the HP-5 capillary column. Interpretation of the mass except Lis C1 and Lis C3 isolates from raw chicken meat. Surprisingly, spectra were conducted using the database of National Institute of DMSO and distilled water extracts of negro pepper elicited non- Standards and Technology (NIST) having more than 62,000 patterns. inhibitory activity against the pathogens at the tested concentration The name, molecular formula, molecular weight, and structure of the (Table 4). components were ascertained. The relative percentage of the chemical In Table 5, the DIZ of ethanolic spice extracts against food-borne constituents in the extracts were expressed as percentage by peak area S. aureus, MRSA, B. cereus, and B. subtilis are shown. The pathogens normalization. responded considerably with different degrees of susceptibility to the extracts with DIZs ranging from 8.3 mm to 16.0 mm. Ethanol extracts of 2.5. Statistical analysis negro pepper showed significant (p < 0.05) zone of inhibition, partic- ularly against S. aureus and MRSA with DIZs of 16 mm and 15.3 mm, Statistical computations for DIZ data were performed using IBM SPSS respectively. Results of antimicrobial activity showed that spice extracts Version 25.0 (SPSS Inc., Chicago, IL, USA). Results were expressed as have varying degrees of effectiveness against bacterial pathogens. means of three replicates ± standard deviation (SD) and subjected to S. aureus and MRSA responded better compared to B. cereus and one-way analysis of variance (ANOVA). Duncan’s Multiple Range Test B. subtilis which demonstrated most resistance to the extracts at the (DMRT) was employed to discriminate between the means whenever tested concentration when disk diffusion assay method was employed. significant difference existed using 5% (p < 0.05) level of confidence. 3.4. Minimum inhibitory concentration (MIC) and minimum bactericidal 3. Results concentration (MBC) 3.1. Prevalence and phenotypic profiles of Listeria spp. in various food Disk diffusion assay may not be a reliable method to conclude the categories antimicrobial activity of spice extract. Thus, MIC and MBC assays were employed to quantitatively evaluate antibacterial activity of extracts to The overall prevalence of Listeria spp. was 23% (13/56) as recorded set up their bacteriostatic and bactericidal concentrations. in Table 1. The prevalence of Listeria spp. was predominantly recorded Using the macro dilution broth and agar dilution methods the ability in raw chicken meat samples (56%, 10/18) compared to RTE (7%, 2/27) of bacterial pathogens to produce visible growth at certain concentra- and dairy products (9%, 1/11). Among the species identified, L. innocua tions was investigated of all spice extracts used in the screening assay. was the most prevalent (18%, 10/56) followed by L. seeligeri (5%, 3/56) The MIC and MBC values as determined by agar and broth dilution and L. marthi (5%, 3/56). L. monocytogenes was prevalent in 7% (4/56) methods respectively are illustrated in Tables 6 and 7. Table 6 shows of the samples with all positive isolates obtained from raw chicken meat. listeriostatic and listericidal concentrations of spice extracts prepared Two unknown isolates (Table 2) recording a prevalence of 4% (2/56) from four different extraction solvents. Of all the different extracts, only were also identified. clove and negro pepper demonstrated ability to suppress the growth of L. monocytogenes with MIC ranging from 0.05% to 0.4% and MBC 3.2. Ethnobotanical information of plant species ranging from 0.1% to > 0.4%. Clove and negro pepper extracts sup- pressed the growth L. monocytogenes mostly at MIC of 0.2%, while 0.4% The ethnobotanical data of utilized spices and their extract pH are (MBC) was adequate to result in total mortality. Moreover, at the same recorded in Table 3. These constitute thirteen plant species among 10 MBC concentration (0.4%), ethanolic clove and negro pepper extracts families representing good diversity of spices used in this study. The did not exert bactericidal effect against L. monocytogenes (Lis C3) isolate medicinal and biological properties of these plant species are evident obtained from raw chicken. Meanwhile, some solvent extracts revealed owing to the diverse use in folkloric medicine and as agents for con- that bactericidal concentrations are enough to elicit a killing effect. For trolling bacterial infections. Various plant extract exhibited varying pH instance, methanol extracts of clove at inhibitory concentration of 0.2% levels ranging from 4.35 to 7.56. demonstrated bactericidal efficacy against L. m. AS-1 (4b) and L. m. Table 1 Prevalence and distribution of Listeria spp. in various food categories. Sample category No. of samples % No. of samples positive for Listeria spp. (%) Total L. monocytogenes L. innocua L. ivanovii L. seeligeri L. marthi Unknown Raw chicken 18 10 (56) 4 (22) 10 (56) 1 (6) 2 (11) 3 (17) 0 (0) 20* RTF 27 2 (7) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (7) 2 Dairy product 11 1 (9) 0 (0) 0 (0) 0 (0) 1 (9) 0 (0) 0 (0) 1 Overall 56 13 (23) 4 (7) 10 (18) 1 (2) 3 (5) 3 (5) 2 (4) 23 * (41) *: repeated number of samples, RTE: Ready-To-Eat food 4 C. Xedzro et al. M i c r o b i o l o g i c a l R e s e a r c h 258 (2022) 126980 Table 2 Phenotypic, biochemical and pathogenicity profiles of isolated Listeria spp. Identified Listeria spp. Phenotypic profile* Biochemical and Sugar fermentation* Pathogenicity profile* MP GR MTB MTA CT VP LR DX MN GL βH CS L. monocytogenes Rod + + + + + + – – + + + L. innocua Rod + + + + + + – – + – – L. ivanovii Rod + + + + + – + – + + – L. seeligeri Rod + + + + + – + – + +w + L. marthi Rod + + + + + – – – + – – Unknown Rod + + + + – – – d d – – Unknown Rod + + + + – – – – d + + *MP: Morphology, GR: Gram’s reaction, MTB: Motility by microscopy, MTA: Motility in semi-solid gar, CT: Catalase test, VP: Voges-Proskauer, LR: L-Rhamnose, DX: D- Xylose, MN: Mannitol, GL: Glucose, βH: β-hemolysis, CS: Chris Atkins Munch Peterson test with S. aureus,+ : positive,-: negative,+ w: weak,d: variable. Table 3 Ethnobotanical classification of utilized spice species and their extract pH. Plant species Family Common name Local Part used Extract Biological action/Medicinal uses Ghanaian pH name Monodora Annonaceae Calabash Wediaba Seed 6.45 Inhibitory effects on fungi (Aspergillus spp., Rhizopus spp.) and bacteria myristica nutmeg (B. Subtilis, S. auereus) (Akise et al., 2020) Treat elevated cholesterol levels ( Nwoso et al., 2015). Piper guineense Piperaceae West African Esrowisa Fruit 7.24 Treat sickle cell anemia (Freiesleben et al., 2015). black pepper Tetrepleura Fabaceae Aidan Prekese Fruit 6.91 Molluscidal activity, anti-convulsant, attenuates high carbohydrate (Kuate tetraptera et al., 2015). Aframomun Zingiberaceae Grains of Fomwisa Fruit/ 5.98 Repellent activity against Sitophilus zeamais, antigrowth against tumor melegueta paradise Seeds cells (Freiesleben et al., 2015; Ukeh et al., 2009). Xylopia aethiopica Annonaceae Negro pepper Hwentia Fruit 5.51 Antiseptic potentials, arrests bleeding, and antifungal efficacies (Konning et al., 2004; Ogbonnia et al., 2008). Inhibitory activity against B. subtilis, S. aureus, Pseudomonas aeruginosa (Fleischer et al., 2008). Pimpinella anisum Apiaceae Aniseed Nketekete Seed 5.43 Treat epilepsy and seizure, antidiabetic, gastro protective (Shojaii and Abdollahi Fard, 2012; Sun et al., 2019). Parkia biglobosa Fabaceae African locust Dawadawa Seed 7.40 Immunomodulating properties (Zou et al., 2014). Inhibitory effects against bean bacteria (Udobi and Onaolapo, 2009) Cinnamomum Lauraceae Cinnamon Cinnamon Bark 4.40 Antimicrobial activity against Helicobacter pylori (Ali et al., 2005) Diabetes, zeylanicum Excessive menstruation and backache (Kim et al., 2006; Lee and Balick, 2005). Piper nigrum Piperaceae Black pepper – Fruit 4.35 Antioxidant, radical scavenging, bactericidal, anti-adhesive activity, treat flu and muscle rushes (Chen et al., 2019; Gülçin, 2005; O’Mahony et al., 2005). Sypzygium Myrtaceae Clove Pepre Flowers/ 5.12 Antibiofilm activity against P. aeruginosa (El-Abed et al., 2011). Antiviral aromaticum Buds activity against HSV-1 and HSV-2 viruses (Kamatou et al., 2012). Capsicum annuum Solanaceae Cayenne Meko Fruit 5.53 Anti-inflammatory and carminative activity (Pandey et al., 2012). Ocimum bacilicum Lamiaceae Basil Akuko besa Leaf 7.56 Antibacterial effect against S. aureus and B. cereus (Adigüzel et al., 2005; Moghaddam et al., 2011). Rauvolfia Apocynaceae Rauvolfia kakapenpen Root 6.85 Treatment of infertility, hypertension, malaria and diarrhea (Lembe et al., vomitoria 2014). Antifilarial activity (Attah et al., 2013). M24–1 (1/2c) strains. Interestingly, the extracts showed similar patterns concentration of negro pepper but required 12xMIC of the extract to of activity against clinical and isolated strains of L. monocytogenes. elicit bactericidal effect. MIC and MBC represented as > 0.4% or > 0.8% Higher concentrations of distilled water extracts (>0.4%) may be indicate that the pathogens did not respond to inhibitory and killing required to elicit inhibitory activity against the test pathogens. Gener- effects, respectively, and that they showed visible colonies on agar plates ally, DMSO and methanol extracts of negro pepper showed strong effi- after 24 h incubation at 37 ◦C. cacy recording MIC as low as 0.05% and 0.1%, respectively, against the In general, negro pepper showed broad activity eliciting relatively pathogens. high inhibitory activities against all the tested food-borne pathogenic In Table 7, antibacterial activity of ethanolic spice extracts expressed bacteria even at the lowest MIC concentration of 0.025% against as MIC and MBC against some common Gram-positive pathogens S. aureus whiles B. cereus, and B. subtilis were the most resistant strains considered in this study are presented. All extracts showed inhibitory (Table 7). Although some extracts showed non-inhibitory activity when effects against S. aureus, except aidan. More importantly, negro pepper disk diffusion method employed, it does not necessarily mean the anti- showed stronger activity against S. aureus, with MIC as low as 0.025% microbial agents were inactive. Agar dilution and broth dilution followed by West African black pepper and African locust bean, with methods proved to be a reliable approach to determine antimicrobial MIC of 0.05%. Methicillin-resistant S. aureus (MRSA) was also suscep- activity. tibility to most of the antimicrobial agents. Of the 13 ethanolic spice extracts, 10 showed considerable effect against MRSA (Table 7). Nevertheless, ethanolic extracts of aidan, African locust bean, and cay- 3.5. Time-kill kinetics enne elicited no bacteriostatic and bactericidal effects against MRSA even at the highest concentration (0.8%) tested. In terms of spore for- The bacteriostatic and bactericidal effects of most active extracts mers, B. cereus and B. subtilis were suppressed at 0.05% bacteriostatic (clove and negro pepper) against cocktail of L. monocytogenes (clinical strains) are illustrated in Fig. 1 (A and B). The results showed that the 5 C. Xedzro et al. M i c r o b i o l o g i c a l R e s e a r c h 258 (2022) 126980 Table 4 extracts exerted strong activity against L. monocytogenes. Concentrations The sensitivity profiles of different solvent spice extracts against equal to 0.5xMIC and 1xMIC demonstrated inhibitory effects against the L. monocytogenes. pathogen showing no significant increase or reduction in original Extraction Spices Diameter of Inhibition Zone: DIZ (mm) inoculum size. Nonetheless, in a time dependent manner the extracts solvent L. m. L. m. Lis C1 Lis C2 Lis C3 Lis C4 demonstrated listericidal effects indicated by total reduction in bacterial AS-1 M24-1 populations at 2xMIC within 10 and 24 h respectively for negro pepper (4b) (1/2c) and clove. Increasing the concentration to 4xMIC resulted in cell death Ethanol Negro 10.0 10.0 10.0 9.7 ± 9.5 ± 9.7 ± within 4 h of more than 3 log units for both extracts. pepper ± 0.0a ± 0.0a ± 0.3a 0.0a 0.3a 0.0a 3.6. Analysis of chemical constituents Clove 12.1 12.2 12.4 12.2 12.0 12.7 ± 0.5b ± 0.4b ± ± ± ± 0.3ab 0.2ab 0.3b 0.3a The detected chemical components present in ethanolic extracts DMSO Negro 8.0 ± 8.0 ± 8.0 ± 8.0 ± 8.0 ± 8.0 ± according to their retention times and elution order on HP-5 MS fused pepper 0.0a 0.0a 0.0a 0.0a 0.0a 0.0a silica capillary column are presented in Table 8. The identification and Clove 11.7 12.0 11.2 10.8 11.0 11.0 characterization established the percent compositions of the major ± 0.5a ± 0.5a ± ± ± ± 0.2a 1.2a 1.4a 1.4a volatile compounds in ethanol extracts. GC-MS analysis revealed three Methanol Negro 9.6 ± 10.3 9.3 ± 9.0 ± 9.8 ± 9.3 ± and eight major bioactive compounds in clove and negro pepper, pepper 0.1bc ± 0.7a 0.1bc 0.0c 0.2ab 0.3bc respectively. The major constituents identified in crude clove extract Clove 12.8 10.8 13.5 12.5 13.3 13.2 were eugenol representing 72% of the total compound, caryophyllene ± ± 0.2d ± ± ± ± bc a b ab ab (10.40%), and phenol, 2-methoxy-4-(2-propenyl)-, acetate (17.15%). 0.5 0.2 0.2 0.3 0.2 Distilled Negro 8.0 ± 8.0 ± 8.0 ± 8.0 ± 8.0 8.0 Identities of the major chemical compounds in crude extracts of negro ± ± water pepper 0.0a 0.0a 0.0a 0.0a 0.0a 0.0a pepper based on the peak area and retention times were (1 R)− 2,6,6- Clove 9.6 ± 11.2 8.0 ± 9.5 ± 8.0 ± 9.5 ± trimethylbicyclo[3.1.1]hept-2-ene (13.58%), β-pinene (42.26%), euca- 0.5a ± 0.7a 0.0a 0.5a 0.0a 0.5a lyptol (9.52%), bicyclo [3.1.1]heptan-3-ol,6,6-dimethyl-2-methylene- L. m AS-1(4b): L. monocytogenes serotype 4b, L. m. M24-1(1/2c): L. mono- (3.86%), octatriene, 1,3-trans-5-trans- (3.93%), 1,6-cyclodecadiene, 1- cytogenes serotype 1/2c, Lis C1, Lis C2, Lis C3 and Lis C4 are L. monocytogenes methyl-5-methylene-8- (1-methylethyl)-, [S-(E,E)]- (10.52%), 2-buta- isolates recovered from raw chicken meat. Different superscripts in the same row none, 3-methoxy-3-methyl- (3.14%), and bicyclo [5.2.0]nonane, 4- indicate response values are statistically different (p < 0.05) methylene-2,8,8-trimethyl-2-vinyl- (13.19%). 4. Discussion Table 5 The sensitivity profiles of ethanolic spice extracts against food-borne pathogens. Though we did not genotypically characterize Listeria isolates, hence Spice species Diameter of Inhibition Zone: DIZ (mm) a limitation to our study, mandatory biochemical tests recommended by S. aureus MRSA B. cereus (IFO B. subtilis Barre et al. (2016) in the capacity of European Union Reference Labo- 209 P 3457) (BF) ratory for Listeria monocytogenes were performed. Hemolysis test, CAMP Calabash nutmeg 8.0 ± 0.0b 8.0 ± 10.7 ± 0.5a 10.7 ± 0.9a test, and sugar fermentation test were additional confirmation test done 0.0b towards identification of Listeria spp. Biochemical identification tests West African black 8.3 ± 0.6b 11.7 ± 8.0 ± 0.0b 8.0 ± 0.0b a were also employed by EI Hag et al. (2020) to confirm Listeria spp. The pepper 0.9 Aidan 8.0 0.0a 8.0 8.0 0.0a 8.0 0.0a phenotypic profiles of the isolates showed consistency with previously ± ± ± ± 0.0a published literature (Barre et al., 2016; den Bakker et al., 2014; Moura Grains of paradise 10.5 ± 0.6a 8.3 ± 8.0 ± 0.0b 8.0 ± 0.0b et al., 2016; Weller et al., 2015). 0.5b The overall prevalence of Listeria spp. was 23% (13/56) which is Negro pepper 16.0 ± 2.0a 15.3 ± 10.2 ± 0.2b 11.2 ± 1.3b a relatively low compared to some other studies that reported higher 0.5 Aniseed 8.0 0.0a 8.0 8.0 0.0a 8.0 0.0a prevalence (Osaili et al., 2011; Şanlıbaba et al., 2018). Meanwhile, ± ± ± ± 0.0a Listeria with pathogenic potential is not commonly reported in Japan. African locust bean 13.9 ± 0.4a 10.0 ± 9.0 ± 1.4b 9.0 ± 1.4b Detection of L. monocytogenes (prevalence of 7%) in our study may pose 0.0b serious threat to the public health. The presence of L. monocytogenes Cinnamon 8.0 ± 0.0a 8.0 ± 8.0 ± 0.0a 8.0 ± 0.0a 0.0a could be attributed to cross contamination which may have occurred Black pepper 8.0 ± 0.0b 10.2 ± 8.0 ± 0.0b 8.0 ± 0.0b during transportation, poor handling, and contamination from pro- 0.2a cessing equipment. Clove 12.7 ± 1.1a 11.2 ± 8.0 ± 0.0b 8.7 ± 0.9b The increasing concern about food safety has triggered the devel- 1.2a a a a opment of abundant natural antimicrobial agents to control and sup-Cayenne 8.0 ± 0.0 8.0 ± 8.0 ± 0.0 8.0 ± 0.0 0.0a press the growth of food-borne and spoilage microorganisms. In ancient Basil 8.0 ± 0.0a 8.0 ± 8.0 ± 0.0a 8.0 ± 0.0a civilization, plant extracts were noteworthy for treating different kinds 0.0a of diseases and nearly 30% accounted for worldwide sales of pharma- Rauvolfia 8.0 ± 0.0a 8.0 ± 8.0 ± 0.0a 8.0 ± 0.0a ceutical products (Soberón et al., 2007). Recently, several potent anti- 0.0a Control (99.5% 8.0 0.0a 8.0 8.0 0.0a 80. 0.0a microbial agents have been used widely to retard microbial growth with ± ± ± ± ethanol) 0.0a particular emphasis and interest in naturally occurring antimicrobials (Egamberdieva et al., 2021). This study demonstrated the ability of some S. aureus 209P: S. aureus stain 209P, MRSA: methicillin-resistant S. aureus, B. plant extracts to suppress the growth of bacterial pathogens when cereus (IFO 3457): B. cereus strain IFO 3457, B. subtilis (BF): B. subtilis strain BF, Different superscripts in the same row indicate response values are statisti- certain concentrations of them were introduced. cally different (p < 0.05). As shown in Tables 4 and 5, the investigated plant species showed antimicrobial efficacies against food-borne pathogens to some extent. Studies have demonstrated that ethanolic extracts of clove, cinnamon, and black pepper were non-inhibitory against S. aureus (Ababutain, 2011) when disk diffusion method was employed. Shan et al. (2007) also 6 C. Xedzro et al. M i c r o b i o l o g i c a l R e s e a r c h 258 (2022) 126980 Table 6 Antibacterial activity of different solvent spice extracts expressed as MIC and MBC (%) against L. monocytogenes. Extraction Solvent Spices L. m. AS-1 (4b) L. m. 24–1 (1/2c) Lis C1 Lis C2 Lis C3 Lis C4 MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC Ethanol Negro pepper 0.2 0.4 0.2 0.2 0.1 0.2 0.2 0.4 0.2 > 0.4 0.2 0.4 Clove 0.2 0.4 0.2 0.4 0.2 0.4 0.2 0.4 0.4 > 0.4 0.2 0.4 DMSO Negro pepper 0.05 0.2 0.05 0.1 0.1 0.2 0.2 0.4 0.2 0.4 0.2 0.4 Clove 0.2 0.4 0.2 0.4 0.2 0.4 0.2 0.4 0.4 0.4 0.2 0.4 Methanol Negro pepper 0.1 0.2 0.1 0.2 0.1 0.2 0.4 0.4 0.4 0.4 0.2 0.4 Clove 0.2 0.2 0.2 0.2 0.2 0.4 0.2 0.4 0.2 0.4 0.2 0.4 Distilled water Negro pepper > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 Clove > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 > 0.4 MIC: minimum inhibitory concentration, MBC: minimum bactericidal concentration. studies, as for instance, Akthar et al. (2014) observed inhibitory po- Table 7 tentials of black pepper against B. cereus, and S. aureus. Akpomie and Antibacterial activity of ethanolic spice extracts expressed as MIC and MBC (%) Ossai (2011) previously reported DIZ value of 9.0 mm for negro pepper against food-borne pathogens. against S. aureus whiles Konning et al. (2004) reported comparable DIZ Spice species S. aureus 209 MRSA B. cereus IFO B. subtilis BF value of 9.8 mm for negro pepper against S. aureus. This study revealed P 3457 higher activity for negro pepper, recording DIZ value of 16.0 mm, MIC MBC MIC MBC MIC MBC MIC MBC compared to previous studies. The different activities of the extracts Calabash 0.4 (-) 0.4 (-) (-) (-) (-) (-) could be ascribed to differences in geographical locations, maturity of nutmeg plant species, time of harvest, methodological differences, and chemo- West African 0.05 0.05 0.05 0.05 0.8 (-) 0.8 (-) type of the extracts. black pepper Hydrophobic properties of natural antimicrobial agents that are Aidan (-) (-) (-) (-) (-) (-) (-) (-) poorly soluble or insoluble in water restricts uniform diffusion through Grains of 0.2 0.4 0.2 0.4 (-) (-) (-) (-) agar media (Klančnik et al., 2010). Thus, disk diffusion test may not be paradise appropriate indicator to determine the antimicrobial activity of plants Negro pepper 0.025 0.05 0.05 0.05 0.05 0.6 0.05 0.6 extracts because the agents are separated and partitioned through the Aniseed 0.4 0.8 0.2 0.8 (-) (-) (-) (-) African locust 0.05 0.4 (-) (-) (-) (-) (-) (-) agar with respect to their water affinities. The method could be ideal and bean appropriate for water soluble antibiotics. Previous reports summarized Cinnamon 0.4 0.8 0.4 (-) (-) (-) (-) (-) that the absence of inhibition zones does not necessarily mean that a Black pepper 0.1 0.6 0.2 0.6 (-) (-) (-) (-) particular agent is inactive especially for non polar compounds which Clove 0.1 0.2 0.1 0.2 (-) (-) (-) (-) Cayenne 0.2 0.6 (-) (-) (-) (-) (-) (-) may not or slowly diffuse in culture media (Klančnik et al., 2010; Basil 0.2 0.4 0.4 (-) (-) (-) (-) (-) Moreno et al., 2006). Also, the size of inhibition zone is not proportional Rauvolfia 0.4 0.6 0.4 (-) (-) (-) (-) (-) to the relative effectiveness of antimicrobial compound. MIC: minimum inhibitory concentration, MBC: minimum bactericidal concen- In the quantitative evaluation in agar and broth assays, we estab- tration, (-): MIC/MBC (>0.8%) or full growth observed. 8% of various solvents lished the MICs and MBCs of the extracts to confirm their antimicrobial were used as control (full growth) potentials at varying test concentrations (0.025% to 0.4%) against Gram-positive bacteria. Extracts of negro pepper and clove showed reported that extract of black pepper had no significant (p > 0.05) ef- similar trend of activity against L. monocytogenes in both qualitative and fects on B. cereus, S. aureus and L. monocytogenes which showed consis- quantitative methods. L. monocytogenes showed susceptibility to only the tency with our results. Furthermore, Adigüzel et al. (2005) reported that two extracts with MIC ranging from 0.1% to > 0.4% (Table 6). To some ethanolic extract of basil was non-inhibitory against B. cereus. None- extent, these results were similar to those of previous studies in which theless there were disparities in the present study compared to other ethanolic extracts of clove were potent against L. monocytogenes (Bayoub et al., 2010; Cui et al., 2018). No comparable literature was obtained for Fig. 1. Dose and time dependent killing curves of ethanolic extracts of negro pepper (A) and clove (B) against cocktail of clinical L. monocytogenes (serogroup 4b and 1/2c). 7 C. Xedzro et al. M i c r o b i o l o g i c a l R e s e a r c h 258 (2022) 126980 Table 8 Major chemical compositions of ethanolic antilisterial extracts. Extract Retention time (min) Name of compounds Molecular formula Composition (%) Negro pepper 4.62 (1 R)− 2,6,6-Trimethylbicyclo [3.1.1] hept-2-ene C10H16 13.58 5.23 β-Pinene C10H16 42.26 6.01 Eucalyptol C10H18O 9.52 7.59 Bicyclo [3.1.1] heptan-3-ol, 6,6-dimethyl-2-methylene- C10H16O 3.86 8.41 Octatriene, 1,3-trans-5-trans- C8H12 3.93 12.29 1,6-Cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-, [S- (E, E)]- C15H24 10.52 17.33 2-Butanone, 3-methoxy-3-methyl- C6H12O2 3.14 21.62 Bicyclo [5.2.0] nonane, 4-methylene-2,8,8-trimethyl-2-vinyl- C15H24 13.19 Clove 10.61 Eugenol C10H12O2 72.45 11.53 Caryophyllene C15H24 10.40 12.72 Phenol, 2-methoxy-4-(2-propenyl)-, acetate C12H14O3 17.15 negro pepper against L. monocytogenes except for Gram-negative bacte- of negro pepper and clove were effective against L. monocytogenes. As ria which were not included in this study. Antimicrobial activity of clove illustrated in Fig. 1, the extracts exerted strong activity against and negro pepper is however evident based on their active compounds. L. monocytogenes resulting in absolute mortality. Concentrations equal to Studies previously done attributed active compounds of clove such as 2xMIC and 4xMIC significantly reduced inoculum size by > 3 log10 eugenol (71.56%), thymol (0.87%), eugenyl acetate (8.99%), car- CFU/mL. Antibacterial agent was regarded listericidal at the lowest yophenyllene oxide (1.67%), nootkatin (1.05%), p-cymene (0.90), concentration that reduced the population size by > 3 log10 CFU/mL guaiol(0.90%), and isolongifolanone (0.86%) to antimicrobial activity (99.9%) and listeriostatic, if the population decreased by 0–3 log10 CFU/ (Nassar et al., 2007). The current study revealed three major chemical mL (Bag et al., 2012; Sharma et al., 2012). compounds of clove (Table 8), eugenol (72.45%), caryophyllene (10.45%), and phenol, 2-methoxy-4-(2-propenyl)-, acetate (17.15%). 5. Conclusions Interestingly, eugenol being the main bioactive component showed consistency with previously published literature. Similarly, Kusalaruk This study revealed that the various plant extracts possess antibac- and Nakano (2021) studied the hurdle effects of plant extract against terial activity. Only clove and negro pepper demonstrated potency E. coli and reported similar chemical compositions of ethanolic clove against L. monocytogenes. Interestingly, negro pepper showed broad extracts. Moreover, another study documented linalool, trans-- spectrum of activity eliciting bacteriostatic efficacy against all the tested β-ocimene, α-farnesene, α-pinene, β-pinene, camphene, and limonene in pathogens. The present findings are promising information that support negro pepper as compounds contributing to antimicrobial activity (Tairu the antibacterial activity of the tested spices as potential candidates to et al., 1999) against bacterial pathogens. Our study revealed eight combat bacterial pathogens associated with food-borne outbreaks. It bioactive components (Table 8) present in negro pepper which may have could also encourage future research on the studies of synergism, ac- conferred inhibitory effects against the tested pathogens. These active tivity in food or food processing systems, and mechanism of activity compounds could penetrate the cell membrane and interact with key against specific pathogens. Furthermore, studies are needed to uncover enzymes resulting in the loss of their functionality and thereby causing pharmaceutical relevance of the tested plant materials and this report impairment of enzymatic mechanisms for energy production and may serve as a steppingstone. To the best of our knowledge, data on the metabolism. Similarly, many of these compounds interfere with phos- antibacterial efficacy of negro pepper against L. monocytogenes is re- pholipid bilayer of the cell membrane and eventually lead to destabli- ported here for the first time. zation of plasma membrane, leakage of intracellular components, severe cell collapse and deformation, and inactivation of genetic materials CRediT authorship contribution statement (Ceruso et al., 2020; Takó et al., 2020). Table 7 illustrates the MICs and MBCs of ethanolic spice extracts All authors have contributed enormously to this manuscript. against S. aureus, MRSA, B. cereus, and B. subtilis. By comparison, the Christian Xedzro designed, planned, and performed the experi- sensitivities of S. aureus and MRSA to the extracts were significantly ments, collected, and analysed the data, wrote the manuscript in higher than those of B. cereus, and B. subtilis. This could be ascribed to consultation with Kwaku Tano-Debrah and Hiroyuki Nakano. Kwaku rapid morphological alterations resulting in significant number of pores, Tano-Debrah devised the project, the main conceptual ideas leading to wrinkled surface, cell protrusions, and aggregation (Clemente et al., this manuscript. This co-author suggested and assisted in getting the 2016; Efenberger-Szmechtyk et al., 2021) and also differences in viru- research samples. Hiroyuki Nakano worked out almost all the technical lent enzymatic response of S. aureus and MRSA to extracts compared to details, and assisted in relevant calculations for the experiment, super- B. cereus, and B. subtilis. Bacillus species were least sensitive due to their vised the project, revised the mauscript, gave technical assistance and resistance to environmental conditions and their ability to form resistant provided research tools and equipment. All authors discussed the re- resting stages known as endospores (Liliwirianis et al., 2011). Poly- sults, commented, and agreed on the manuscript. phenolic compounds embedded in plant extracts are involved in multi- ple mode of action such as inhibiting protein and ATP synthesis and thereby causing changes in metabolic processes and inhibiting DNA Funding synthesis (Efenberger-Szmechtyk et al., 2021) which could lead to cellular instability. Furthermore, it was noted that the bacteriostatic and This research was supported and funded by the Japanese Ministry of bactericidal results of some test antimicrobial agents had MIC values Education, Culture, Sports, Science and Technology (MEXT) and Hir- equal to their MBCs. This explains the fact that these agents at such oshima University. concentrations exert inhibitory effect against bacterial proliferation characterized by mortality induction. 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