Anti-bacterial activity of essential oils against multidrug-resistant foodborne pathogens isolated from raw milk

Atividade antibacteriana de óleos essenciais contra patógenos alimentares multirresistentes isolados de leite cru

M. Yasir A. Nawaz S. Ghazanfar M. K. Okla A. Chaudhary Wahidah H. Al M. N. Ajmal H. AbdElgawad Z. Ahmad F. Abbas A. Wadood Z. Manzoor N. Akhtar M. Din Y. Hameed M. Imran About the authors

Abstract

The presence of pathogenic bacteria in food is considered as a primary cause of food-borne illness and food quality deterioration worldwide. The present study aimed to determine the effectiveness of five essential oils (EOs) against multidrug-resistant foodborne pathogens. In the current study Gram-negative bacteria (Escherichia, Enterobacter, Citrobacter, Proteus, Pseudomonas, and Klebsiella) and the Gram-positive bacteria Staphylococcus were isolated from raw milk and biochemically characterized. The anti-bacterial effect of different antibiotics and EOs (thyme, oregano, lemongrass, mint, and rosemary) was determined using the standard disc diffusion method. The antibiogram study revealed that Gram-negative bacteria were highly resistant to penicillin while Staphylococcus was resistant to streptomycin, amoxicillin, and lincomycin. Moderate resistance was observed to doxycycline, amikacin, enrofloxacin, kanamycin and cefixime. Isolates were found less resistant to gentamycin, chloramphenicol, and ciprofloxacin. EOs showed a broad range of antimicrobial activity against all bacteria except P. aeruginosa. Of these, thyme was more effective against most of the multi-drug resistant bacterial strains and formed the largest zone of inhibition (26 mm) against Escherichia followed by oregano oil (18 mm) against Staphylococcus (p<0.05). Klebsiella spp and Citrobacter spp showed resistance to mint and lemongrass oil respectively. The EOs such as lemongrass, mint and rosemary were less active against all the bacteria. The findings of the recent study suggest the use of EOs as natural antibacterial agents for food preservation.

Keywords:
pathogenic bacteria; zone of inhibition; antibacterial agents; lemongrass oil; aldehydes

Resumo

A presença de bactérias patogênicas em alimentos é considerada a principal causa de doenças transmitidas por alimentos e deterioração da qualidade dos alimentos em todo o mundo. O presente estudo teve como objetivo determinar a eficácia de cinco óleos essenciais (OEs) contra patógenos de origem alimentar multirresistentes. No presente estudo, bactérias Gram-negativas (Escherichia, Enterobacter, Citrobacter, Proteus, Pseudomonas e Klebsiella) e as bactérias Gram-positivas Staphylococcus foram isoladas do leite cru e caracterizadas bioquimicamente. O efeito antibacteriano de diferentes antibióticos e OEs (tomilho, orégano, capim-limão, hortelã e alecrim) foi determinado usando o método padrão de difusão em disco. O estudo do antibiograma revelou que as bactérias Gram-negativas eram altamente resistentes à penicilina, enquanto o Staphylococcus era resistente à estreptomicina, amoxicilina e lincomicina. Foi observada resistência moderada à doxiciclina, amicacina, enrofloxacina, canamicina e cefixima. Os isolados foram encontrados menos resistentes à gentamicina, cloranfenicol e ciprofloxacina. Os OEs mostraram uma ampla gama de atividade antimicrobiana contra todas as bactérias, exceto P. aeruginosa. Destes, o tomilho foi mais eficaz contra a maioria das cepas bacterianas multirresistentes e formou a maior zona de inibição (26 mm) contra Escherichia seguido de óleo de orégano (18 mm) contra Staphylococcus (p<0,05). Klebsiella spp e Citrobacter spp apresentaram resistência ao óleo de menta e capim-limão, respectivamente. Os OEs como capim-limão, hortelã e alecrim foram menos ativos contra todas as bactérias. Os resultados do estudo recente sugerem o uso de OEs como agentes antibacterianos naturais para conservação de alimentos.

Palavras-chave:
bactérias patogênicas; zona de inibição; agentes antibacterianos; óleo de capim-limão; aldeídos

1. Introduction

Milk and milk-related products are good sources of nutrition for humans. However, its high water, protein and vitamin contents, and neutral pH provide excellent conditions for microbial growth which can happen in raw, pasteurized, or refrigerated milk. Bacteria significantly affect milk quality and its quantity by releasing toxins which increases the risk of food poisoning and infections (Bytyqi et al., 2013BYTYQI, H., VEHAPI, I., REXHEPI, S., THAQI, M., SALLAHI, D. and MEHMETI, I., 2013. Impact of bacterial and somatic cells content on quality fresh milk in small-scale dairy farms in Kosovo. Food and Nutrition Sciences, vol. 4, no. 10, pp. 1014-1020. http://dx.doi.org/10.4236/fns.2013.410132.
http://dx.doi.org/10.4236/fns.2013.41013...
; Hernández-Cortez et al., 2017HERNÁNDEZ-CORTEZ, C., PALMA-MARTÍNEZ, I., GONZALEZ-AVILA, L.U., GUERRERO-MANDUJANO, A., COLMENERO SOLÍS, R. and CASTRO-ESCARPULLI, G., 2017. Food poisoning caused by bacteria (food toxins). In: N. MALANGU, ed. Poisoning: from specific toxic agents to novel rapid and simplified techniques for analysis. London: IntechOpen.; Mankai et al., 2012MANKAI, M., BOULARES, M., BEN MOUSSA, O., KAROUI, R. and HASSOUNA, M., 2012. The effect of refrigerated storage of raw milk on the physicochemical and microbiological quality of Tunisian semihard Gouda‐type cheese during ripening. International Journal of Dairy Technology, vol. 65, no. 2, pp. 250-259. http://dx.doi.org/10.1111/j.1471-0307.2012.00822.x.
http://dx.doi.org/10.1111/j.1471-0307.20...
). Many of these illnesses are caused by Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes and Escherichia coli (toxin-producing) (Addis and Sisay, 2015ADDIS, M. and SISAY, D., 2015. A review on major food borne bacterial illnesses. Journal of Tropical Diseases & Public Health, vol. 3, no. 4, pp. 176-183.; Bintsis, 2017BINTSIS, T., 2017. Foodborne pathogens. AIMS Microbiol, vol. 3, no. 3, pp. 529-563. http://dx.doi.org/10.3934/microbiol.2017.3.529. PMid:31294175.
http://dx.doi.org/10.3934/microbiol.2017...
; Havelaar et al., 2015HAVELAAR, A.H., KIRK, M.D., TORGERSON, P.R., GIBB, H.J., HALD, T., LAKE, R.J., PRAET, N., BELLINGER, D.C., DE SILVA, N.R., GARGOURI, N., SPEYBROECK, N., CAWTHORNE, A., MATHERS, C., STEIN, C., ANGULO, F.J. and DEVLEESSCHAUWER, B., 2015. World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Medicine, vol. 12, no. 12, e1001923. http://dx.doi.org/10.1371/journal.pmed.1001923. PMid:26633896.
http://dx.doi.org/10.1371/journal.pmed.1...
). These bacteria interact with antibiotics which can be present in the food system and develop resistance. Antibiotic resistance among foodborne pathogens is a worldwide problem. This problem arises due to the extensive use of antimicrobial feed additives for therapeutic purposes and as a growth enhancer for animal production (Baynes et al., 2016BAYNES, R.E., DEDONDER, K., KISSELL, L., MZYK, D., MARMULAK, T., SMITH, G., TELL, L., GEHRING, R., DAVIS, J. and RIVIERE, J.E., 2016. Health concerns and management of select veterinary drug residues. Food and Chemical Toxicology, vol. 88, pp. 112-122. http://dx.doi.org/10.1016/j.fct.2015.12.020. PMid:26751035.
http://dx.doi.org/10.1016/j.fct.2015.12....
; Manyi-Loh et al., 2018MANYI-LOH, C., MAMPHWELI, S., MEYER, E. and OKOH, A., 2018. Antibiotic use in agriculture and its consequential resistance in environmental sources: potential public health implications. Molecules, vol. 23, no. 4, pp. 795. http://dx.doi.org/10.3390/molecules23040795. PMid:29601469.
http://dx.doi.org/10.3390/molecules23040...
; Moyane et al., 2013MOYANE, J., JIDEANI, A. and AIYEGORO, O., 2013. Antibiotics usage in food-producing animals in South Africa and impact on human: antibiotic resistance. African Journal of Microbiological Research, vol. 7, no. 24, pp. 2990-2997. http://dx.doi.org/10.5897/AJMR2013.5631.
http://dx.doi.org/10.5897/AJMR2013.5631...
). With the increase of drug resistance, the efficacy of several drugs and antimicrobial agents has been reduced significantly. Therefore, regulatory authorities which monitor the food and beverage industries urge the use of natural food preservatives. Essential oils (EOs) are aromatic oily materials that can be extracted from different parts of the plant. Their chemical composition often includes terpenoids (specifically monoterpenes and sesquiterpenes) and low molecular weight compounds (alcohols, aldehydes, ketones, lactones, acetyls, oxides, esters and phenols). EOs are known to have antibacterial, antifungal, antiviral, anticancer and antioxidant effects (Fitsiou and Pappa, 2019FITSIOU, E. and PAPPA, A., 2019. Anticancer activity of essential oils and other extracts from aromatic plants grown in Greece. Antioxidants, vol. 8, no. 8, pp. 290. http://dx.doi.org/10.3390/antiox8080290. PMid:31394842.
http://dx.doi.org/10.3390/antiox8080290...
; Helal et al., 2019HELAL, I.M., EL-BESSOUMY, A., AL-BATAINEH, E., JOSEPH, M.R.P., RAJAGOPALAN, P., CHANDRAMOORTHY, H.C. and BEN HADJ AHMED, S., 2019. Antimicrobial efficiency of essential oils from traditional medicinal plants of Asir Region, Saudi Arabia, over drug resistant isolates. BioMed Research International, vol. 2019, pp. 8928306. http://dx.doi.org/10.1155/2019/8928306. PMid:30792999.
http://dx.doi.org/10.1155/2019/8928306...
; Nadjib, 2020NADJIB, B.M., 2020. Effective antiviral activity of essential oils and their characteristic terpenes against coronaviruses: an update. Journal of Pharmacology & Clinical Toxicology, vol. 8, pp. 1138.; Wińska et al., 2019WIŃSKA, K., MĄCZKA, W., ŁYCZKO, J., GRABARCZYK, M., CZUBASZEK, A. and SZUMNY, A., 2019. Essential oils as antimicrobial agents: myth or real alternative? Molecules, vol. 24, no. 11, pp. 2130. http://dx.doi.org/10.3390/molecules24112130. PMid:31195752.
http://dx.doi.org/10.3390/molecules24112...
). Moreover, they are non-toxic to humans and the environment and are believed to have limited chances for the development of resistance in bacteria (Chouhan et al., 2017CHOUHAN, S., SHARMA, K. and GULERIA, S., 2017. Antimicrobial activity of some essential oils-present status and future perspectives. Medicines, vol. 4, no. 3, pp. 58. http://dx.doi.org/10.3390/medicines4030058. PMid:28930272.
http://dx.doi.org/10.3390/medicines40300...
; Yap et al., 2014YAP, P.S.X., YIAP, B.C., PING, H.C. and LIM, S.H.E., 2014. Essential oils, a new horizon in combating bacterial antibiotic resistance. The Open Microbiology Journal, vol. 8, no. 1, pp. 6-14. http://dx.doi.org/10.2174/1874285801408010006. PMid:24627729.
http://dx.doi.org/10.2174/18742858014080...
). The antibacterial activity (in vitro and food assays) of EOs have been reported against several food borne pathogens such as L. monocytogenes, Staphylococcus, S. enterica, E. coil. Pseudomonas. aeruginosa and Candida albicans (Mittal et al., 2019MITTAL, R.P., RANA, A. and JAITAK, V., 2019. Essential oils: an impending substitute of synthetic antimicrobial agents to overcome antimicrobial resistance. Current Drug Targets, vol. 20, no. 6, pp. 605-624. http://dx.doi.org/10.2174/1389450119666181031122917. PMid:30378496.
http://dx.doi.org/10.2174/13894501196661...
; Puškárová et al., 2017PUŠKÁROVÁ, A., BUČKOVÁ, M., KRAKOVÁ, L., PANGALLO, D. and KOZICS, K., 2017. The antibacterial and antifungal activity of six essential oils and their cyto/genotoxicity to human HEL 12469 cells. Scientific Reports, vol. 7, no. 1, pp. 8211. http://dx.doi.org/10.1038/s41598-017-08673-9. PMid:28811611.
http://dx.doi.org/10.1038/s41598-017-086...
; Santos et al., 2017SANTOS, M.I.S., MARTINS, S.R., VERÍSSIMO, C.S.C., NUNES, M.J.C., LIMA, A.I.G., FERREIRA, R.M.S.B., PEDROSO, L., SOUSA, I. and FERREIRA, M.A.S.S., 2017. Essential oils as antibacterial agents against food-borne pathogens: are they really as useful as they are claimed to be? Journal of Food Science and Technology, vol. 54, no. 13, pp. 4344-4352. http://dx.doi.org/10.1007/s13197-017-2905-0. PMid:29184240.
http://dx.doi.org/10.1007/s13197-017-290...
). Some EOs can control food fermentation by controlling Lactobacillus plantarum and Saccharomyces cerevisiae growth (Liu et al., 2017LIU, Q., MENG, X., LI, Y., ZHAO, C.-N., TANG, G.-Y. and LI, H.-B., 2017. Antibacterial and antifungal activities of spices. International Journal of Molecular Sciences, vol. 18, no. 6, pp. 1283. http://dx.doi.org/10.3390/ijms18061283. PMid:28621716.
http://dx.doi.org/10.3390/ijms18061283...
; Mith et al., 2014MITH, H., DURÉ, R., DELCENSERIE, V., ZHIRI, A., DAUBE, G. and CLINQUART, A., 2014. Antimicrobial activities of commercial essential oils and their components against food-borne pathogens and food spoilage bacteria. Food Science & Nutrition, vol. 2, no. 4, pp. 403-416. http://dx.doi.org/10.1002/fsn3.116. PMid:25473498.
http://dx.doi.org/10.1002/fsn3.116...
). Their bactericidal mechanisms include hydrophobic interactions with bacterial cell envelopes, coagulating of membrane protein and destruction of membrane potential which result in cellular death (Chouhan et al., 2017CHOUHAN, S., SHARMA, K. and GULERIA, S., 2017. Antimicrobial activity of some essential oils-present status and future perspectives. Medicines, vol. 4, no. 3, pp. 58. http://dx.doi.org/10.3390/medicines4030058. PMid:28930272.
http://dx.doi.org/10.3390/medicines40300...
). Although the antibacterial activity of EOs has been reported against large numbers of foodborne pathogens, there are limited reports on the activity of EOs against multi drug-resistant (MDR) foodborne pathogens. The present study aimed to determine the activity of five EOs against MDR bacteria isolated from raw milk from Quetta valley of Baluchistan.

2. Materials and Methods

2.1. Study area and sampling

The study was carried out at Center for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta, Pakistan. Geographically representative milk samples (n=48) from different markets of north, south, east and west regions of Quetta city were collected during January 10 to January 18, 2022. Samples (12 each from the four regions) were collected into sterile glass bottles and transported to the Microbiology laboratory at the Center for Advanced Study in Vaccinology and Biotechnology (CASVAB) via a cold chain for bacterial analysis.

2.2. Isolation and purification of bacteria

For isolation of the Gram-negative bacterial strains, milk samples (0.01 mL) were spread aseptically on different selective and differential media i.e., Eosine Methylene blue agar (LAB, Heywood, UK) for Escherichia, MacConkey agar (LAB, Lancashire, UK) for coliform and Proteus spp, and Cetrimide agar (Oxoid, Basingstoke, UK) for Pseudomonas spp. They were further identified through biochemical tests including indole production (Oxoid, Basingstoke, UK), glucose metabolism with the methyl red (Merck, Darmstadt, Germany), acetone production using Voges proskauer (Merck, Darmstadt Germany), the simmon citrate utilization (BBL, Sparks, MD, USA), triple sugar iron (LAB, Heywood, UK), catalase, oxidase, urease and motility tests. Moreover, coagulase test, catalase test, indole production, methyl red test, Voges-proskauer reaction, urease production, citrate utilization were also utilized to identify Staphylococcus. In addition, for isolating of Staphylococcus, the loop full (0.01 mL) of the milk sample was spread on mannitol salt agar (MSA) (Oxoid, Basingstoke, UK) and incubated aerobically at 37°C for 24 h. The isolated bacteria were preserved in Brain Heart Infusion broth (Oxoid, Basingstoke, UK) with the addition of 30% glycerol at -70°C for further experiments.

2.3. Antibiotic sensitivity assay

The antibiogram assay used the disc diffusion method. Bacterial strains were dispensed in tubes containing 5 mL normal saline and the turbidity was adjusted to a McFarland turbidity standard of 0.5 yielding 1.5 × “108” CFU/mL. Cotton swabs were soaked and spread onto the Muller Hinton Agar ((MHA), Oxoid, UK). The antibiotic discs (Oxoid, Basingstoke, UK) amikacin (30µg), amoxicillin (30µg), cefixime (5µg), chloramphenicol (5µg), ciprofloxacin (5µg), doxycycline (30µg), enrofloxacin (5µg), gentamycin (10µg), lincomycin (10µg), kanamycin (30µg), methicillin (10µg), penicillin G (10 Unit), streptomycin (10µg), sulphamethoxazole trimethoprim (25µg), tetracycline (30µg), and vancomycin (30µg) were used and aerobically incubated at 37 oC for 24 hours in an inverted position. After incubation, the antibiotic sensitivity and resistance pattern of each strain was studied. The clear zone around each disc was measured. The results were recorded according to the Clinical and Laboratory Standards Institute (Weinstein and Lewis II, 2020WEINSTEIN, M.P. and LEWIS II, J.S., 2020. The clinical and laboratory standards institute subcommittee on antimicrobial susceptibility testing: background, organization, functions, and processes. Journal of Clinical Microbiology, vol. 58, no. 3, pp. e01864-e01819. http://dx.doi.org/10.1128/JCM.01864-19. PMid:31915289.
http://dx.doi.org/10.1128/JCM.01864-19...
).

2.4. Selection of plants

Five herbal plants including Thyme (Thymus vulgaris), Oregano (Oregano vulgare), Rosemary (Rosemarinus officinalis), Mint (Mentha spicata), and Lemongrass (Cymbopogon citratus) were selected to investigate their antimicrobial potential. All these plants were collected from Arid Zone Research Center (AZRC) Quetta, Pakistan.

2.5. Extraction of essential oils

Fresh ariel leaves were collected from plants and chopped into pieces. Approximately 250 g leaves of each plant were used for steam distillation in a Clevenger type instrument for three (3) hours. A light denser layer of oil developed in a burette at the surface of the water and was carefully separated and the yield was calculated. Extracted EOs were stored at 4oC in a sealed vial covered with aluminium foil till further use (Baj et al., 2015BAJ, T., SIENIAWSKA, E., KOWALSKI, R., WESOLOWSKP, M. and ULEWICZ-MAGULSKA, B., 2015. Effectiveness of the deryng and clevenger-type apparatus in isolation of various types of components of essential oil from the Mutelina purpurea Thell. flowers. Acta Poloniae Pharmaceutica, vol. 72, no. 3, pp. 507-515. PMid:26642659.).

2.6. Antibacterial assay of essential oils

Three antibiotic-resistant strains of each bacterial species were selected for this assay. The antibacterial effect of EOs in the liquid phase was analyzed through the disc diffusion method 25. As described above 100 μL of 0.5 McFarland (yielding “108” CFU/mL) were applied to the Muller Hinton Agar (Oxoid) plates. A pre-sterilized Whatman filter paper (6 mm diameter) was then placed on the plates. Afterwards, 10 μL of each EO was added to the Whatman filter paper. The plates were allowed to dry for 30 to 60 minutes at room temperature under aseptic conditions and then incubated in an inverted position at 37 °C for 24 h. After incubation, the antibacterial activity of EOs in the form of the clear zone (in millimetre) around the disc was measured (Fernandez-Lopez et al., 2005FERNANDEZ-LOPEZ, P., SANCHEZ, C., BATLLE, R. and NERIN, C., 2005. Solid and vapour phase antimicrobial activities of six essential oils: susceptibility of selected food borne bacterial and fungal strains. Journal of Agricultural and Food Chemistry, vol. 53, no. 17, pp. 6939-6946. http://dx.doi.org/10.1021/jf050709v. PMid:16104824.
http://dx.doi.org/10.1021/jf050709v...
).

2.7. Statistical analysis

Data with three repeated measurements (antimicrobial activity of oils) was analyzed and calculated using the one-way ANOVA test setting the p< 0.05.

3. Results

3.1. Incidence of pathogenic bacteria in raw milk

Bacterial species including Escherichia, Enterobacter, Citrobacter, Proteus, Pseudomonas, and Klebsiella and Staphylococcus were identified in the raw milk samples. Among all the isolates, Escherichia was the most abundant contaminant of raw milk with a 27% prevalence. The prevalence of Escherichia, Enterobacter, Citrobacter, Proteus, Pseudomonas, and Klebsiella and Staphylococcus species were approximately 18%, 15%, 13%, 13%, 13%, 10%, and 4% respectively (Table 1).

Table 1
The incidence of pathogens in milk samples.

3.2. Antibiotic resistance pattern

Variable trends of antibiotic resistance were shown by both types of (Gram-negative and Gram-positive) bacteria identified from raw milk (Figure 1). For example, Escherichia was resistant to amoxicillin, penicillin G, streptomycin (100%), lincomycin (62%), kanamycin (46%), doxycycline and sulfamethoxazole-trimethoprim (31%). While, less resistance of Escherichia was also observed to cefixime (23%), chloramphenicol, gentamycin, and tetracycline (15% each), and enrofloxacin (8%). Moreover, all the strains of Escherichia were sensitive to ciprofloxacin (Figure 1). It is previously acknowledged that in Gram-negative bacteria, different factors like a change in hydrophobic properties or mutations in porins can create the MDR (Breijyeh et al., 2020BREIJYEH, Z., JUBEH, B. and KARAMAN, R., 2020. Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules, vol. 25, no. 6, pp. 1340. http://dx.doi.org/10.3390/molecules25061340. PMid:32187986.
http://dx.doi.org/10.3390/molecules25061...
), therefore, might be due to this reason, here in our study, the Gram-negative bacteria showed the maximum MDR.

Figure 1
This graph depicts the percentage antibiotics resistance of bacterial isolateed from raw milk samples. X-axis = percentage of antibiotic resistence, and Y-axis = names of antibiotics.

3.3. Antibacterial efficacies of essential oils against drug-resistant strains

The highest yield of rosemary (Rosemarinus officinalis, 0.89%) followed by lemongrass (Cymbopogon citratus, 0.80%), oregano (Oregano vulgare, 0.78%), mint (Mentha spicata, 0.71%) and thyme (Thymus vulgaris, 0.67%) plants were determined (Table 2).

Table 2
The yields of essential oils (v/m) from different plants using steam distillation process.

EOs were categorized as being effective based on previously published study by Celikel and Kavas (2008)CELIKEL, N. and KAVAS, G., 2008. Antimicrobial properties of some essential oils against some pathogenic microorganisms. Czech Journal of Food Sciences, vol. 26, no. 3, pp. 174-181. http://dx.doi.org/10.17221/1603-CJFS.
http://dx.doi.org/10.17221/1603-CJFS...
. According to which, if the total diameter was <8 mm the organism was resistant, inhibition zones of 9 to 14 mm sensitive (+), and 15 to 19 mm very sensitive (++), for diameter > 20 mm extremely sensitive (+++).

Thymus vulgaris (thyme) was most active among all tested EOs and formed the largest zone of inhibition of 26 mm against Escherichia that was significantly higher (p<0.05) than any other EOs zones of inhibition. The zone of inhibition against the other bacteria Enterobacter spp, Staphylococcus, Citrobacter spp, Proteus spp, and Klebsiella spp, were 21, 20, 20, 18 and 13 mm respectively (Figure 2). The oregano oil formed the largest zone of inhibition against Staphylococcus (18 mm) and smallest against Klebsiella spp (12 mm). Oregano oil gave a significantly (p<0.05) larger zone of inhibition against Enterobacter spp, compared to lemon, mint, or rosemary oils. The Cymbopogon citratus (lemongrass) oil showed moderate activity. Lemongrass oil was more effective against Staphylococcus (15.1 mm zone of inhibition) than the Gram-negative bacteria. Figure 3 represent the zone of inhibition formed by lemongrass, thyme, and mint oils against Staphylococcus. The Rosemary officinalis (rosemary) and Mentha spicata (mint) oils gave the lowest antibacterial activity. The highest zone of inhibition for Rosemary officinalis (rosemary) oil was 10.4 mm against Enterobacter and Proteus spp, and then reduced to 9 mm against Citrobacter spp. Mint oil had the largest zone of inhibition (14 mm) against Enterobacter spp and the smallest zone of inhibition (10.4 mm) against Escherichia.

Figure 2
Mean inhibition zones of different essential oils against different strains of Escherichia, Enterobacter spp, Citrobacter spp, Proteus spp, Pseudomonas spp and Staphylococcus, and Klebsiella spp through disc diffusion method (Results are mean of three independent experiments). X-axis = zone of inhibition in mm, and Y-axis = names of pathogens.
Figure 3
This figure depicts the inhibition of Staphylococcus by thyme, mint and lemongrass oils through disc diffusion method. Footnote: where T, thyme; M, mint; and L.G, lemongrass.

4. Discussion

Milk is known to be an excellent medium for microbial growth (Abdul Khalil et al., 2014ABDUL KHALIL, K., MUSTAFA, S., MOHAMMAD, R., BIN ARIFF, A., SHAARI, Y., ABDUL MANAP, Y., AHMAD, S.A. and DAHALAN, F.A., 2014. Optimization of milk-based medium for efficient cultivation of Bifidobacterium pseudocatenulatum G4 using face-centered central composite-response surface methodology. BioMed Research International, vol. 2014, pp. 787989. http://dx.doi.org/10.1155/2014/787989. PMid:24527457.
http://dx.doi.org/10.1155/2014/787989...
). Bacteria may gain access to the milk from different sources including, workers, the animal's normal microbiota, the farm environment, and utensils and cause a variety of milk-borne diseases (Ahmedsham et al., 2018AHMEDSHAM, M., AMZA, N. and TAMIRU, M., 2018. Review on milk and milk product safety, quality assurance and control. International Journal of Livestock Production, vol. 9, no. 4, pp. 67-78. http://dx.doi.org/10.5897/IJLP2017.0403.
http://dx.doi.org/10.5897/IJLP2017.0403...
; Berhe et al., 2020BERHE, G., WASIHUN, A.G., KASSAYE, E. and GEBRESELASIE, K., 2020. Milk-borne bacterial health hazards in milk produced for commercial purpose in Tigray, northern Ethiopia. BMC Public Health, vol. 20, no. 1, pp. 894. http://dx.doi.org/10.1186/s12889-020-09016-6. PMid:32517771.
http://dx.doi.org/10.1186/s12889-020-090...
). In the current study, several human pathogens were detected in raw milk. Some strains of these bacteria were found resistant to commercially antibiotics. In the present study, Escherichia was the most abundant contaminant of raw milk (27%), but other bacteria were also present. Similar bacteria with slightly different rates such as Escherichia 32% Enterobacter spp 29%, Klebsiella spp 19%, and Citrobacter spp 1% were also detected in raw milk in an earlier study (Salman and Hamad, 2011SALMAN, A.M. and HAMAD, I.M., 2011. Enumeration and identification of coliform bacteria from raw milk in Khartoum State, Sudan. Journal of Cell and Animal Biology, vol. 5, pp. 121-128.). A high rate of isolation of Escherichia is a sign of the poor quality of milk, unhygienic conditions in the farm, and milk collection and processing (Iqbal et al., 2004IQBAL, M., KHAN, M.A., DARAZ, B. and SIDDIQUE, U., 2004. Bacteriology of mastitic milk and in vitro antibiogram of the isolates. Pakistan Veterinary Journal, vol. 24, no. 4, pp. 161-164.). Escherichia in the milk is the major sign of faecal contamination, which may enter the milk through animal faeces or by the unwashed hands of milkmen (Islam et al., 2018ISLAM, M.A., ROY, S., NABI, A., SOLAIMAN, S., RAHMAN, M., HUQ, M., SIDDIQUEE, N.A. and AHMED, N., 2018. Microbiological quality assessment of milk at different stages of the dairy value chain in a developing country setting. International Journal of Food Microbiology, vol. 278, pp. 11-19. http://dx.doi.org/10.1016/j.ijfoodmicro.2018.04.028. PMid:29689333.
http://dx.doi.org/10.1016/j.ijfoodmicro....
). Similarly, the high incidence of Gram-negative bacteria like Enterobacter, Citrobacter, Proteus and Pseudomonas spp may also point out the poor sanitation and hygiene (Drzewiecka, 2016DRZEWIECKA, D., 2016. Significance and roles of Proteus spp. bacteria in natural environments. Microbial Ecology, vol. 72, no. 4, pp. 741-758. http://dx.doi.org/10.1007/s00248-015-0720-6. PMid:26748500.
http://dx.doi.org/10.1007/s00248-015-072...
; Garedew et al., 2012GAREDEW, L., BERHANU, A., MENGESHA, D. and TSEGAY, G., 2012. Identification of gram-negative bacteria from critical control points of raw and pasteurized cow milk consumed at Gondar town and its suburbs, Ethiopia. BMC Public Health, vol. 12, no. 1, pp. 950-950. http://dx.doi.org/10.1186/1471-2458-12-950. PMid:23131015.
http://dx.doi.org/10.1186/1471-2458-12-9...
). Similar to other bacteria, Staphylococcus in raw milk may hurt public health as enterotoxins produced by Staphylococcus are heat stable and can survive at pasteurization (McMillan et al., 2016MCMILLAN, K., MOORE, S.C., MCAULEY, C.M., FEGAN, N. and FOX, E.M., 2016. Characterization of Staphylococcus aureus isolates from raw milk sources in Victoria, Australia. BMC Microbiology, vol. 16, no. 1, pp. 169. http://dx.doi.org/10.1186/s12866-016-0789-1. PMid:27473328.
http://dx.doi.org/10.1186/s12866-016-078...
). Milk can be contaminated with Staphylococcus directly from the animal's udder during causes of mastitis (Cobirka et al., 2020COBIRKA, M., TANCIN, V. and SLAMA, P., 2020. Epidemiology and classification of mastitis. Animals, vol. 10, no. 12, pp. 2212. http://dx.doi.org/10.3390/ani10122212. PMid:33255907.
http://dx.doi.org/10.3390/ani10122212...
; Petersson-Wolfe et al., 2010PETERSSON-WOLFE, C.S., MULLARKY, I.K. and JONES, G.M., 2010. Staphylococcus aureus mastitis: cause, detection, and control. Blacksburg: VirginiaTech.).

The indiscriminate use of antimicrobials leads to drug resistance which threatens the health of both animals and humans. Antimicrobial resistance in food animals has a significant impact on animal health and may be associated with resistant infections in humans (Ma et al., 2021MA, F., XU, S., TANG, Z., LI, Z. and ZHANG, L., 2021. Use of antimicrobials in food animals and impact of transmission of antimicrobial resistance on humans. Biosafety and Health, vol. 3, no. 1, pp. 32-38. http://dx.doi.org/10.1016/j.bsheal.2020.09.004.
http://dx.doi.org/10.1016/j.bsheal.2020....
). In our study, several isolates have shown a multi-drug resistant (MDR) pattern. Gram-negative bacteria had higher resistance to penicillin G 100%, streptomycin 91%, amoxicillin 85%, lincomycin 72%, sulfamethoxazole-trimethoprim 43%, and enrofloxacin 39%. Multidrug-resistant bacteria such as Escherichia, Klebsiella, Enterobacter, Proteus vulgaris and Staphylococcus species have been detected in raw milk previously (Mahami et al., 2011MAHAMI, T., ODONKOR, S., YARO, M. and ADU-GYAMFI, A., 2011. Prevalence of antibiotic resistant bacteria in milk sold in Accra. International Research Journal of Microbiology, vol. 2, pp. 126-132.). According to Koluman and Dikici (Koluman and Dikici, 2013KOLUMAN, A. and DIKICI, A., 2013. Antimicrobial resistance of emerging foodborne pathogens: status quo and global trends. Critical Reviews in Microbiology, vol. 39, no. 1, pp. 57-69. http://dx.doi.org/10.3109/1040841X.2012.691458. PMid:22639875.
http://dx.doi.org/10.3109/1040841X.2012....
) several food born human pathogens were resistant to tetracycline and chloramphenicol but in the present work tetracycline and chloramphenicol were highly effective antibiotics and generally low level of resistance was observed. The low resistance towards chloramphenicol might be because its use is rare at local dairy farms. The isolated bacteria including Escherichia, Enterobacter, Citrobacter, Proteus, Pseudomonas, and Klebsiella and Staphylococcus have also shown MDR against different antibiotics. Moreover, the antibacterial activity of five EOs were evaluated against these bacteria. All the EOs were found effective against Gram-positive as well as Gram-negative bacteria. EOs are hydrophobic in nature so can easily make partition in the lipids of the bacterial cell membrane, resulting disturbing the structures and rendering cell membrane more permeable to leak out K+ and ATP (Lopez-Romero et al., 2015LOPEZ-ROMERO, J.C., GONZÁLEZ-RÍOS, H., BORGES, A. and SIMÕES, M., 2015. Antibacterial effects and mode of action of selected essential oils components against Escherichia coli and Staphylococcus aureus. Evidence-Based Complementary and Alternative Medicine, vol. 2015, pp. 795435. http://dx.doi.org/10.1155/2015/795435. PMid:26221178.
http://dx.doi.org/10.1155/2015/795435...
; Sikkema et al., 1994SIKKEMA, J., DE BONT, J.A. and POOLMAN, B., 1994. Interactions of cyclic hydrocarbons with biological membranes. The Journal of Biological Chemistry, vol. 269, no. 11, pp. 8022-8028. http://dx.doi.org/10.1016/S0021-9258(17)37154-5. PMid:8132524.
http://dx.doi.org/10.1016/S0021-9258(17)...
). Probably, their mechanism of action is therefore be similar to other phenolic compounds which involves disturbance of the cytoplasmic membrane, the proton motive force (PMF), electron flow, active transport and coagulation of cell contents (Chouhan et al., 2017CHOUHAN, S., SHARMA, K. and GULERIA, S., 2017. Antimicrobial activity of some essential oils-present status and future perspectives. Medicines, vol. 4, no. 3, pp. 58. http://dx.doi.org/10.3390/medicines4030058. PMid:28930272.
http://dx.doi.org/10.3390/medicines40300...
). Resistance among Pseudomonas spp to the various EOs has been reported earlier (El-Hosseiny et al., 2014EL-HOSSEINY, L., EL-SHENAWY, M., HAROUN, M. and ABDULLAH, F., 2014. Comparative evaluation of the inhibitory effect of some essential oils with antibiotics against Pseudomonas aeruginosa. International Journal of Antibiotics, vol. 2014, pp. 586252. http://dx.doi.org/10.1155/2014/586252.
http://dx.doi.org/10.1155/2014/586252...
). Pseudomonas spp poses a serious resistance to EOs could be due to possession of restrictive outer membrane barrier (Mann et al., 2000MANN, C., COX, S. and MARKHAM, J., 2000. The outer membrane of Pseudomonas aeruginosa NCTC 6749 contributes to its tolerance to the essential oil of Melaleuca alternifolia (tea tree oil). Letters in Applied Microbiology, vol. 30, no. 4, pp. 294-297. http://dx.doi.org/10.1046/j.1472-765x.2000.00712.x. PMid:10792649.
http://dx.doi.org/10.1046/j.1472-765x.20...
) and the presence of specific lipopolysaccharides in their cell wall (Nostro et al., 2000NOSTRO, A., GERMANO, M., D’ANGELO, V., MARINO, A. and CANNATELLI, M., 2000. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity. Letters in Applied Microbiology, vol. 30, no. 5, pp. 379-384. http://dx.doi.org/10.1046/j.1472-765x.2000.00731.x. PMid:10792667.
http://dx.doi.org/10.1046/j.1472-765x.20...
). But how P. aeruginosa resist EOs to work against it, needs to be explored in future.

Thyme had strong antimicrobial activity against Escherichia followed by oregano oil. Such effect of both the EOs was also investigated previously (Mith et al., 2014MITH, H., DURÉ, R., DELCENSERIE, V., ZHIRI, A., DAUBE, G. and CLINQUART, A., 2014. Antimicrobial activities of commercial essential oils and their components against food-borne pathogens and food spoilage bacteria. Food Science & Nutrition, vol. 2, no. 4, pp. 403-416. http://dx.doi.org/10.1002/fsn3.116. PMid:25473498.
http://dx.doi.org/10.1002/fsn3.116...
; Puškárová et al., 2017PUŠKÁROVÁ, A., BUČKOVÁ, M., KRAKOVÁ, L., PANGALLO, D. and KOZICS, K., 2017. The antibacterial and antifungal activity of six essential oils and their cyto/genotoxicity to human HEL 12469 cells. Scientific Reports, vol. 7, no. 1, pp. 8211. http://dx.doi.org/10.1038/s41598-017-08673-9. PMid:28811611.
http://dx.doi.org/10.1038/s41598-017-086...
). Our finding did not match with earlier reports where thyme was effective against P. aeruginosa but remained ineffective towards Escherichia (Nascimento et al., 2000NASCIMENTO, G.G.F., LOCATELLI, J., FREITAS, P.C. and SILVA, G.L., 2000. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian Journal of Microbiology, vol. 31, no. 4, pp. 247-256. http://dx.doi.org/10.1590/S1517-83822000000400003.
http://dx.doi.org/10.1590/S1517-83822000...
). High antimicrobial action of thyme and oregano species have been attributed to the presence of phenolic compounds e.g., thymol and carvacrol (Gavanji et al., 2015GAVANJI, S., MOHAMMADI, E., LARKI, B. and BAKHTARI, A., 2015. Chemical composition identification and comparison antibacterial activity of some essential oils against three pathogenic bacteria. International Journal of Scientific Research in Knowledge, vol. 3, no. 4, pp. 94-105. http://dx.doi.org/10.12983/ijsrk-2015-p0094-0105.
http://dx.doi.org/10.12983/ijsrk-2015-p0...
; Swamy et al., 2016SWAMY, M.K., AKHTAR, M.S. and SINNIAH, U.R., 2016. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: an updated review. Evidence-Based Complementary and Alternative Medicine, vol. 2016, pp. 3012462. http://dx.doi.org/10.1155/2016/3012462. PMid:28090211.
http://dx.doi.org/10.1155/2016/3012462...
) and p-cymene or γ-terpinene (Simirgiotis et al., 2020SIMIRGIOTIS, M.J., BURTON, D., PARRA, F., LÓPEZ, J., MUÑOZ, P., ESCOBAR, H. and PARRA, C., 2020. Antioxidant and antibacterial capacities of Origanum vulgare L. essential oil from the arid andean region of chile and its chemical characterization by GC-MS. Metabolites, vol. 10, no. 10, pp. 414. http://dx.doi.org/10.3390/metabo10100414. PMid:33081116.
http://dx.doi.org/10.3390/metabo10100414...
). Individual inhibitory effects of thymol and carvacrol against Gram-positive e.g., Staphylococcus and Gram-negative bacteria e.g., P. aeruginosa, Escherichia, K. pneumonia was also reported previously (Fadli et al., 2011FADLI, M., CHEVALIER, J., SAAD, A., MEZRIOUI, N.E., HASSANI, L. and PAGES, J.M., 2011. Essential oils from Moroccan plants as potential chemosensitisers restoring antibiotic activity in resistant Gram-negative bacteria. International Journal of Antimicrobial Agents, vol. 38, no. 4, pp. 325-330. http://dx.doi.org/10.1016/j.ijantimicag.2011.05.005. PMid:21752605.
http://dx.doi.org/10.1016/j.ijantimicag....
; Gavanji et al., 2015GAVANJI, S., MOHAMMADI, E., LARKI, B. and BAKHTARI, A., 2015. Chemical composition identification and comparison antibacterial activity of some essential oils against three pathogenic bacteria. International Journal of Scientific Research in Knowledge, vol. 3, no. 4, pp. 94-105. http://dx.doi.org/10.12983/ijsrk-2015-p0094-0105.
http://dx.doi.org/10.12983/ijsrk-2015-p0...
; Rosato et al., 2010ROSATO, A., PIARULLI, M., CORBO, F., MURAGLIA, M., CARONE, A., VITALI, M.E. and VITALI, C., 2010. In vitro synergistic antibacterial action of certain combinations of gentamicin and essential oils. Current Medicinal Chemistry, vol. 17, no. 28, pp. 3289-3295. http://dx.doi.org/10.2174/092986710792231996. PMid:20666717.
http://dx.doi.org/10.2174/09298671079223...
). There is a possibility that these constitutes work together against bacteria. There were different active compounds in the oil extracts as different sizes of zones were seen. It may indicate that when they are together, as likely near the filter paper, they act in synergy to inhibit the growth (Figure 3) which needs to be tested in future studies.

Rosemary oil produced the smallest zone of inhibition compared to other EOs when tested at the same concentration. Variation in zones of inhibition might be due to the presence of different active compounds such as α-pinene, boranyl acetate, camphor, 1,8 cineole in rosemary EO (Tomi et al., 2016TOMI, K., KITAO, M., KONISHI, N., MURAKAMI, H., MATSUMURA, Y. and HAYASHI, T., 2016. Enantioselective GC-MS analysis of volatile components from rosemary (Rosmarinus officinalis L.) essential oils and hydrosols. Bioscience, Biotechnology, and Biochemistry, vol. 80, no. 5, pp. 840-847. http://dx.doi.org/10.1080/09168451.2016.1146066. PMid:26923429.
http://dx.doi.org/10.1080/09168451.2016....
). Lemongrass oil showed strong antibacterial activity against Staphylococcus than Gram-negative bacteria. All the tested isolates were susceptible to lemongrass oil except Citrobacter and Pseudomonas spp. A relatively higher zone of inhibition (15 mm) was observed against Staphylococcus followed by Proteus spp. Moderate inhibition was observed with all the other experimental strains. These findings are supported by previous study (Wannissorn et al., 2005WANNISSORN, B., JARIKASEM, S., SIRIWANGCHAI, T. and THUBTHIMTHED, S., 2005. Antibacterial properties of essential oils from Thai medicinal plants. Fitoterapia, vol. 76, no. 2, pp. 233-236. http://dx.doi.org/10.1016/j.fitote.2004.12.009. PMid:15752638.
http://dx.doi.org/10.1016/j.fitote.2004....
). A larger zone of inhibition (28 mm) of lemongrass oil was reported against Staphylococcus in a previous study (Silveira et al., 2012SILVEIRA, S.M., CUNHA JÚNIOR, A., SCHEUERMANN, G.N., SECCHI, F.L. and VIEIRA, C.R.W., 2012. Chemical composition and antimicrobial activity of essential oils from selected herbs cultivated in the South of Brazil against food spoilage and foodborne pathogens. Ciência Rural, vol. 42, no. 7, pp. 1300-1306. http://dx.doi.org/10.1590/S0103-84782012000700026.
http://dx.doi.org/10.1590/S0103-84782012...
) but the inhibition areas to all the other isolates were similar to recent findings. A larger zone of inhibition may be due to the presence of several active compounds in lemongrass oil such as citral, limonene, neral, geranial, citronellal, and neryl acetate (Ugbabe et al., 2016UGBABE, G.E., OKHALE, S.E., EGHAREVBA, H.O. and IBRAHIM, J.A., 2016. Foliar microscopy and GC-MS analysis of the volatile oil constituents of the leaf of Cymbopogon citratus (DC.) Stapt. (Poaceae/Graminae). International Journal of Basic and Applied Sciences, vol. 5, pp. 45-49.). Spearmint oil was found active against both Gram-positive and Gram-negative bacteria except Pseudomonas and Klebsiella spp which were resistant. In similar studies, significant inhibition (15 mm) against Escherichia was noted with spearmint oil (Lixandru et al., 2010LIXANDRU, B.E., DRĂCEA, N.O., DRAGOMIRESCU, C.C., DRĂGULESCU, E.C., COLDEA, I.L., ANTON, L., DOBRE, E., ROVINARU, C. and CODIŢĂ, I., 2010. Antimicrobial activity of plant essential oils against bacterial and fungal species involved in food poisoning and/or food decay. Roumanian Archives of Microbiology and Immunology, vol. 69, no. 4, pp. 224-230. PMid:21462837.; Sulieman et al., 2011SULIEMAN, A.M.E., ABDELRAHMAN, S.E. and ABDEL RAHIM, A., 2011. Phytochemical analysis of local spearmint (Mentha spicata) leaves and detection of the antimicrobial activity of its oil. Journal of Microbiology Research, vol. 1, no. 1, pp. 1-4. http://dx.doi.org/10.5923/j.microbiology.20110101.01.
http://dx.doi.org/10.5923/j.microbiology...
). In another study, Gram-negative bacteria e.g., Escherichia, Pseudomonas spp and Enterobacter spp were resistant but Staphylococcus and Proteus spp were susceptible to spearmint oil (Silveira et al., 2012SILVEIRA, S.M., CUNHA JÚNIOR, A., SCHEUERMANN, G.N., SECCHI, F.L. and VIEIRA, C.R.W., 2012. Chemical composition and antimicrobial activity of essential oils from selected herbs cultivated in the South of Brazil against food spoilage and foodborne pathogens. Ciência Rural, vol. 42, no. 7, pp. 1300-1306. http://dx.doi.org/10.1590/S0103-84782012000700026.
http://dx.doi.org/10.1590/S0103-84782012...
). These findings differ from a recent study as Escherichia, and Enterobacter spp were sensitive to spearmint oil in the current study. Giving the differences in the activity of spearmint oil might be due to differences in bioactive constituents such as β-myrcene, limonene, 1,8-cineole and menthone (Benabdallah et al., 2018BENABDALLAH, A., BOUMENDJEL, M., AISSI, O., RAHMOUNE, C., BOUSSAID, M. and MESSAOUD, C., 2018. Chemical composition, antioxidant activity and acetylcholinesterase inhibitory of wild Mentha species from northeastern Algeria. South African Journal of Botany, vol. 116, pp. 131-139. http://dx.doi.org/10.1016/j.sajb.2018.03.002.
http://dx.doi.org/10.1016/j.sajb.2018.03...
; Snoussi et al., 2015SNOUSSI, M., NOUMI, E., TRABELSI, N., FLAMINI, G., PAPETTI, A. and DE FEO, V., 2015. Mentha spicata essential oil: chemical composition, antioxidant and antibacterial activities against planktonic and biofilm cultures of Vibrio spp. strains. Molecules, vol. 20, no. 8, pp. 14402-14424. http://dx.doi.org/10.3390/molecules200814402. PMid:26262604.
http://dx.doi.org/10.3390/molecules20081...
).

The diameter of inhibition zones formed by EOs varied in different studies. This could be due to many factors, firstly, the climatic and environmental conditions which bring changes in the composition of EOs (Janssen et al., 1987JANSSEN, A., SCHEFFER, J. and SVENDSEN, A.B., 1987. Antimicrobial activity of essential oils: a 1976-1986 literature review. Aspects of the test methods. Planta Medica, vol. 53, no. 5, pp. 395-398. http://dx.doi.org/10.1055/s-2006-962755. PMid:3324126.
http://dx.doi.org/10.1055/s-2006-962755...
; Sivropoulou et al., 1995SIVROPOULOU, A., KOKKINI, S., LANARAS, T. and ARSENAKIS, M., 1995. Antimicrobial activity of mint essential oils. Journal of Agricultural and Food Chemistry, vol. 43, no. 9, pp. 2384-2388. http://dx.doi.org/10.1021/jf00057a013.
http://dx.doi.org/10.1021/jf00057a013...
). This suggested that there may be a difference in the susceptibility of strains isolated from different sites or a difference in active compounds of EOs extracted from different geographical regions. Secondly, the technique used to evaluate the antimicrobial potential of EOs, and the selection of test organisms differs from previous experiments. Also tests oils with the same common name may be derived from different plant species.

Acknowledgments

The authors extend their appreciation to the researchers supporting project no (RSP-2021/293) at King Saud University, Riyadh, Saudi Arabia.

References

  • ABDUL KHALIL, K., MUSTAFA, S., MOHAMMAD, R., BIN ARIFF, A., SHAARI, Y., ABDUL MANAP, Y., AHMAD, S.A. and DAHALAN, F.A., 2014. Optimization of milk-based medium for efficient cultivation of Bifidobacterium pseudocatenulatum G4 using face-centered central composite-response surface methodology. BioMed Research International, vol. 2014, pp. 787989. http://dx.doi.org/10.1155/2014/787989 PMid:24527457.
    » http://dx.doi.org/10.1155/2014/787989
  • ADDIS, M. and SISAY, D., 2015. A review on major food borne bacterial illnesses. Journal of Tropical Diseases & Public Health, vol. 3, no. 4, pp. 176-183.
  • AHMEDSHAM, M., AMZA, N. and TAMIRU, M., 2018. Review on milk and milk product safety, quality assurance and control. International Journal of Livestock Production, vol. 9, no. 4, pp. 67-78. http://dx.doi.org/10.5897/IJLP2017.0403
    » http://dx.doi.org/10.5897/IJLP2017.0403
  • BAJ, T., SIENIAWSKA, E., KOWALSKI, R., WESOLOWSKP, M. and ULEWICZ-MAGULSKA, B., 2015. Effectiveness of the deryng and clevenger-type apparatus in isolation of various types of components of essential oil from the Mutelina purpurea Thell. flowers. Acta Poloniae Pharmaceutica, vol. 72, no. 3, pp. 507-515. PMid:26642659.
  • BAYNES, R.E., DEDONDER, K., KISSELL, L., MZYK, D., MARMULAK, T., SMITH, G., TELL, L., GEHRING, R., DAVIS, J. and RIVIERE, J.E., 2016. Health concerns and management of select veterinary drug residues. Food and Chemical Toxicology, vol. 88, pp. 112-122. http://dx.doi.org/10.1016/j.fct.2015.12.020 PMid:26751035.
    » http://dx.doi.org/10.1016/j.fct.2015.12.020
  • BENABDALLAH, A., BOUMENDJEL, M., AISSI, O., RAHMOUNE, C., BOUSSAID, M. and MESSAOUD, C., 2018. Chemical composition, antioxidant activity and acetylcholinesterase inhibitory of wild Mentha species from northeastern Algeria. South African Journal of Botany, vol. 116, pp. 131-139. http://dx.doi.org/10.1016/j.sajb.2018.03.002
    » http://dx.doi.org/10.1016/j.sajb.2018.03.002
  • BERHE, G., WASIHUN, A.G., KASSAYE, E. and GEBRESELASIE, K., 2020. Milk-borne bacterial health hazards in milk produced for commercial purpose in Tigray, northern Ethiopia. BMC Public Health, vol. 20, no. 1, pp. 894. http://dx.doi.org/10.1186/s12889-020-09016-6 PMid:32517771.
    » http://dx.doi.org/10.1186/s12889-020-09016-6
  • BINTSIS, T., 2017. Foodborne pathogens. AIMS Microbiol, vol. 3, no. 3, pp. 529-563. http://dx.doi.org/10.3934/microbiol.2017.3.529 PMid:31294175.
    » http://dx.doi.org/10.3934/microbiol.2017.3.529
  • BREIJYEH, Z., JUBEH, B. and KARAMAN, R., 2020. Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules, vol. 25, no. 6, pp. 1340. http://dx.doi.org/10.3390/molecules25061340 PMid:32187986.
    » http://dx.doi.org/10.3390/molecules25061340
  • BYTYQI, H., VEHAPI, I., REXHEPI, S., THAQI, M., SALLAHI, D. and MEHMETI, I., 2013. Impact of bacterial and somatic cells content on quality fresh milk in small-scale dairy farms in Kosovo. Food and Nutrition Sciences, vol. 4, no. 10, pp. 1014-1020. http://dx.doi.org/10.4236/fns.2013.410132
    » http://dx.doi.org/10.4236/fns.2013.410132
  • CELIKEL, N. and KAVAS, G., 2008. Antimicrobial properties of some essential oils against some pathogenic microorganisms. Czech Journal of Food Sciences, vol. 26, no. 3, pp. 174-181. http://dx.doi.org/10.17221/1603-CJFS
    » http://dx.doi.org/10.17221/1603-CJFS
  • CHOUHAN, S., SHARMA, K. and GULERIA, S., 2017. Antimicrobial activity of some essential oils-present status and future perspectives. Medicines, vol. 4, no. 3, pp. 58. http://dx.doi.org/10.3390/medicines4030058 PMid:28930272.
    » http://dx.doi.org/10.3390/medicines4030058
  • COBIRKA, M., TANCIN, V. and SLAMA, P., 2020. Epidemiology and classification of mastitis. Animals, vol. 10, no. 12, pp. 2212. http://dx.doi.org/10.3390/ani10122212 PMid:33255907.
    » http://dx.doi.org/10.3390/ani10122212
  • DRZEWIECKA, D., 2016. Significance and roles of Proteus spp. bacteria in natural environments. Microbial Ecology, vol. 72, no. 4, pp. 741-758. http://dx.doi.org/10.1007/s00248-015-0720-6 PMid:26748500.
    » http://dx.doi.org/10.1007/s00248-015-0720-6
  • EL-HOSSEINY, L., EL-SHENAWY, M., HAROUN, M. and ABDULLAH, F., 2014. Comparative evaluation of the inhibitory effect of some essential oils with antibiotics against Pseudomonas aeruginosa International Journal of Antibiotics, vol. 2014, pp. 586252. http://dx.doi.org/10.1155/2014/586252
    » http://dx.doi.org/10.1155/2014/586252
  • FADLI, M., CHEVALIER, J., SAAD, A., MEZRIOUI, N.E., HASSANI, L. and PAGES, J.M., 2011. Essential oils from Moroccan plants as potential chemosensitisers restoring antibiotic activity in resistant Gram-negative bacteria. International Journal of Antimicrobial Agents, vol. 38, no. 4, pp. 325-330. http://dx.doi.org/10.1016/j.ijantimicag.2011.05.005 PMid:21752605.
    » http://dx.doi.org/10.1016/j.ijantimicag.2011.05.005
  • FERNANDEZ-LOPEZ, P., SANCHEZ, C., BATLLE, R. and NERIN, C., 2005. Solid and vapour phase antimicrobial activities of six essential oils: susceptibility of selected food borne bacterial and fungal strains. Journal of Agricultural and Food Chemistry, vol. 53, no. 17, pp. 6939-6946. http://dx.doi.org/10.1021/jf050709v PMid:16104824.
    » http://dx.doi.org/10.1021/jf050709v
  • FITSIOU, E. and PAPPA, A., 2019. Anticancer activity of essential oils and other extracts from aromatic plants grown in Greece. Antioxidants, vol. 8, no. 8, pp. 290. http://dx.doi.org/10.3390/antiox8080290 PMid:31394842.
    » http://dx.doi.org/10.3390/antiox8080290
  • GAREDEW, L., BERHANU, A., MENGESHA, D. and TSEGAY, G., 2012. Identification of gram-negative bacteria from critical control points of raw and pasteurized cow milk consumed at Gondar town and its suburbs, Ethiopia. BMC Public Health, vol. 12, no. 1, pp. 950-950. http://dx.doi.org/10.1186/1471-2458-12-950 PMid:23131015.
    » http://dx.doi.org/10.1186/1471-2458-12-950
  • GAVANJI, S., MOHAMMADI, E., LARKI, B. and BAKHTARI, A., 2015. Chemical composition identification and comparison antibacterial activity of some essential oils against three pathogenic bacteria. International Journal of Scientific Research in Knowledge, vol. 3, no. 4, pp. 94-105. http://dx.doi.org/10.12983/ijsrk-2015-p0094-0105
    » http://dx.doi.org/10.12983/ijsrk-2015-p0094-0105
  • HAVELAAR, A.H., KIRK, M.D., TORGERSON, P.R., GIBB, H.J., HALD, T., LAKE, R.J., PRAET, N., BELLINGER, D.C., DE SILVA, N.R., GARGOURI, N., SPEYBROECK, N., CAWTHORNE, A., MATHERS, C., STEIN, C., ANGULO, F.J. and DEVLEESSCHAUWER, B., 2015. World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Medicine, vol. 12, no. 12, e1001923. http://dx.doi.org/10.1371/journal.pmed.1001923 PMid:26633896.
    » http://dx.doi.org/10.1371/journal.pmed.1001923
  • HELAL, I.M., EL-BESSOUMY, A., AL-BATAINEH, E., JOSEPH, M.R.P., RAJAGOPALAN, P., CHANDRAMOORTHY, H.C. and BEN HADJ AHMED, S., 2019. Antimicrobial efficiency of essential oils from traditional medicinal plants of Asir Region, Saudi Arabia, over drug resistant isolates. BioMed Research International, vol. 2019, pp. 8928306. http://dx.doi.org/10.1155/2019/8928306 PMid:30792999.
    » http://dx.doi.org/10.1155/2019/8928306
  • HERNÁNDEZ-CORTEZ, C., PALMA-MARTÍNEZ, I., GONZALEZ-AVILA, L.U., GUERRERO-MANDUJANO, A., COLMENERO SOLÍS, R. and CASTRO-ESCARPULLI, G., 2017. Food poisoning caused by bacteria (food toxins). In: N. MALANGU, ed. Poisoning: from specific toxic agents to novel rapid and simplified techniques for analysis London: IntechOpen.
  • IQBAL, M., KHAN, M.A., DARAZ, B. and SIDDIQUE, U., 2004. Bacteriology of mastitic milk and in vitro antibiogram of the isolates. Pakistan Veterinary Journal, vol. 24, no. 4, pp. 161-164.
  • ISLAM, M.A., ROY, S., NABI, A., SOLAIMAN, S., RAHMAN, M., HUQ, M., SIDDIQUEE, N.A. and AHMED, N., 2018. Microbiological quality assessment of milk at different stages of the dairy value chain in a developing country setting. International Journal of Food Microbiology, vol. 278, pp. 11-19. http://dx.doi.org/10.1016/j.ijfoodmicro.2018.04.028 PMid:29689333.
    » http://dx.doi.org/10.1016/j.ijfoodmicro.2018.04.028
  • JANSSEN, A., SCHEFFER, J. and SVENDSEN, A.B., 1987. Antimicrobial activity of essential oils: a 1976-1986 literature review. Aspects of the test methods. Planta Medica, vol. 53, no. 5, pp. 395-398. http://dx.doi.org/10.1055/s-2006-962755 PMid:3324126.
    » http://dx.doi.org/10.1055/s-2006-962755
  • KOLUMAN, A. and DIKICI, A., 2013. Antimicrobial resistance of emerging foodborne pathogens: status quo and global trends. Critical Reviews in Microbiology, vol. 39, no. 1, pp. 57-69. http://dx.doi.org/10.3109/1040841X.2012.691458 PMid:22639875.
    » http://dx.doi.org/10.3109/1040841X.2012.691458
  • LIU, Q., MENG, X., LI, Y., ZHAO, C.-N., TANG, G.-Y. and LI, H.-B., 2017. Antibacterial and antifungal activities of spices. International Journal of Molecular Sciences, vol. 18, no. 6, pp. 1283. http://dx.doi.org/10.3390/ijms18061283 PMid:28621716.
    » http://dx.doi.org/10.3390/ijms18061283
  • LIXANDRU, B.E., DRĂCEA, N.O., DRAGOMIRESCU, C.C., DRĂGULESCU, E.C., COLDEA, I.L., ANTON, L., DOBRE, E., ROVINARU, C. and CODIŢĂ, I., 2010. Antimicrobial activity of plant essential oils against bacterial and fungal species involved in food poisoning and/or food decay. Roumanian Archives of Microbiology and Immunology, vol. 69, no. 4, pp. 224-230. PMid:21462837.
  • LOPEZ-ROMERO, J.C., GONZÁLEZ-RÍOS, H., BORGES, A. and SIMÕES, M., 2015. Antibacterial effects and mode of action of selected essential oils components against Escherichia coli and Staphylococcus aureus Evidence-Based Complementary and Alternative Medicine, vol. 2015, pp. 795435. http://dx.doi.org/10.1155/2015/795435 PMid:26221178.
    » http://dx.doi.org/10.1155/2015/795435
  • MA, F., XU, S., TANG, Z., LI, Z. and ZHANG, L., 2021. Use of antimicrobials in food animals and impact of transmission of antimicrobial resistance on humans. Biosafety and Health, vol. 3, no. 1, pp. 32-38. http://dx.doi.org/10.1016/j.bsheal.2020.09.004
    » http://dx.doi.org/10.1016/j.bsheal.2020.09.004
  • MAHAMI, T., ODONKOR, S., YARO, M. and ADU-GYAMFI, A., 2011. Prevalence of antibiotic resistant bacteria in milk sold in Accra. International Research Journal of Microbiology, vol. 2, pp. 126-132.
  • MANKAI, M., BOULARES, M., BEN MOUSSA, O., KAROUI, R. and HASSOUNA, M., 2012. The effect of refrigerated storage of raw milk on the physicochemical and microbiological quality of Tunisian semihard Gouda‐type cheese during ripening. International Journal of Dairy Technology, vol. 65, no. 2, pp. 250-259. http://dx.doi.org/10.1111/j.1471-0307.2012.00822.x
    » http://dx.doi.org/10.1111/j.1471-0307.2012.00822.x
  • MANN, C., COX, S. and MARKHAM, J., 2000. The outer membrane of Pseudomonas aeruginosa NCTC 6749 contributes to its tolerance to the essential oil of Melaleuca alternifolia (tea tree oil). Letters in Applied Microbiology, vol. 30, no. 4, pp. 294-297. http://dx.doi.org/10.1046/j.1472-765x.2000.00712.x PMid:10792649.
    » http://dx.doi.org/10.1046/j.1472-765x.2000.00712.x
  • MANYI-LOH, C., MAMPHWELI, S., MEYER, E. and OKOH, A., 2018. Antibiotic use in agriculture and its consequential resistance in environmental sources: potential public health implications. Molecules, vol. 23, no. 4, pp. 795. http://dx.doi.org/10.3390/molecules23040795 PMid:29601469.
    » http://dx.doi.org/10.3390/molecules23040795
  • MCMILLAN, K., MOORE, S.C., MCAULEY, C.M., FEGAN, N. and FOX, E.M., 2016. Characterization of Staphylococcus aureus isolates from raw milk sources in Victoria, Australia. BMC Microbiology, vol. 16, no. 1, pp. 169. http://dx.doi.org/10.1186/s12866-016-0789-1 PMid:27473328.
    » http://dx.doi.org/10.1186/s12866-016-0789-1
  • MITH, H., DURÉ, R., DELCENSERIE, V., ZHIRI, A., DAUBE, G. and CLINQUART, A., 2014. Antimicrobial activities of commercial essential oils and their components against food-borne pathogens and food spoilage bacteria. Food Science & Nutrition, vol. 2, no. 4, pp. 403-416. http://dx.doi.org/10.1002/fsn3.116 PMid:25473498.
    » http://dx.doi.org/10.1002/fsn3.116
  • MITTAL, R.P., RANA, A. and JAITAK, V., 2019. Essential oils: an impending substitute of synthetic antimicrobial agents to overcome antimicrobial resistance. Current Drug Targets, vol. 20, no. 6, pp. 605-624. http://dx.doi.org/10.2174/1389450119666181031122917 PMid:30378496.
    » http://dx.doi.org/10.2174/1389450119666181031122917
  • MOYANE, J., JIDEANI, A. and AIYEGORO, O., 2013. Antibiotics usage in food-producing animals in South Africa and impact on human: antibiotic resistance. African Journal of Microbiological Research, vol. 7, no. 24, pp. 2990-2997. http://dx.doi.org/10.5897/AJMR2013.5631
    » http://dx.doi.org/10.5897/AJMR2013.5631
  • NADJIB, B.M., 2020. Effective antiviral activity of essential oils and their characteristic terpenes against coronaviruses: an update. Journal of Pharmacology & Clinical Toxicology, vol. 8, pp. 1138.
  • NASCIMENTO, G.G.F., LOCATELLI, J., FREITAS, P.C. and SILVA, G.L., 2000. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian Journal of Microbiology, vol. 31, no. 4, pp. 247-256. http://dx.doi.org/10.1590/S1517-83822000000400003
    » http://dx.doi.org/10.1590/S1517-83822000000400003
  • NOSTRO, A., GERMANO, M., D’ANGELO, V., MARINO, A. and CANNATELLI, M., 2000. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity. Letters in Applied Microbiology, vol. 30, no. 5, pp. 379-384. http://dx.doi.org/10.1046/j.1472-765x.2000.00731.x PMid:10792667.
    » http://dx.doi.org/10.1046/j.1472-765x.2000.00731.x
  • PETERSSON-WOLFE, C.S., MULLARKY, I.K. and JONES, G.M., 2010. Staphylococcus aureus mastitis: cause, detection, and control Blacksburg: VirginiaTech.
  • PUŠKÁROVÁ, A., BUČKOVÁ, M., KRAKOVÁ, L., PANGALLO, D. and KOZICS, K., 2017. The antibacterial and antifungal activity of six essential oils and their cyto/genotoxicity to human HEL 12469 cells. Scientific Reports, vol. 7, no. 1, pp. 8211. http://dx.doi.org/10.1038/s41598-017-08673-9 PMid:28811611.
    » http://dx.doi.org/10.1038/s41598-017-08673-9
  • ROSATO, A., PIARULLI, M., CORBO, F., MURAGLIA, M., CARONE, A., VITALI, M.E. and VITALI, C., 2010. In vitro synergistic antibacterial action of certain combinations of gentamicin and essential oils. Current Medicinal Chemistry, vol. 17, no. 28, pp. 3289-3295. http://dx.doi.org/10.2174/092986710792231996 PMid:20666717.
    » http://dx.doi.org/10.2174/092986710792231996
  • SALMAN, A.M. and HAMAD, I.M., 2011. Enumeration and identification of coliform bacteria from raw milk in Khartoum State, Sudan. Journal of Cell and Animal Biology, vol. 5, pp. 121-128.
  • SANTOS, M.I.S., MARTINS, S.R., VERÍSSIMO, C.S.C., NUNES, M.J.C., LIMA, A.I.G., FERREIRA, R.M.S.B., PEDROSO, L., SOUSA, I. and FERREIRA, M.A.S.S., 2017. Essential oils as antibacterial agents against food-borne pathogens: are they really as useful as they are claimed to be? Journal of Food Science and Technology, vol. 54, no. 13, pp. 4344-4352. http://dx.doi.org/10.1007/s13197-017-2905-0 PMid:29184240.
    » http://dx.doi.org/10.1007/s13197-017-2905-0
  • SIKKEMA, J., DE BONT, J.A. and POOLMAN, B., 1994. Interactions of cyclic hydrocarbons with biological membranes. The Journal of Biological Chemistry, vol. 269, no. 11, pp. 8022-8028. http://dx.doi.org/10.1016/S0021-9258(17)37154-5 PMid:8132524.
    » http://dx.doi.org/10.1016/S0021-9258(17)37154-5
  • SILVEIRA, S.M., CUNHA JÚNIOR, A., SCHEUERMANN, G.N., SECCHI, F.L. and VIEIRA, C.R.W., 2012. Chemical composition and antimicrobial activity of essential oils from selected herbs cultivated in the South of Brazil against food spoilage and foodborne pathogens. Ciência Rural, vol. 42, no. 7, pp. 1300-1306. http://dx.doi.org/10.1590/S0103-84782012000700026
    » http://dx.doi.org/10.1590/S0103-84782012000700026
  • SIMIRGIOTIS, M.J., BURTON, D., PARRA, F., LÓPEZ, J., MUÑOZ, P., ESCOBAR, H. and PARRA, C., 2020. Antioxidant and antibacterial capacities of Origanum vulgare L. essential oil from the arid andean region of chile and its chemical characterization by GC-MS. Metabolites, vol. 10, no. 10, pp. 414. http://dx.doi.org/10.3390/metabo10100414 PMid:33081116.
    » http://dx.doi.org/10.3390/metabo10100414
  • SIVROPOULOU, A., KOKKINI, S., LANARAS, T. and ARSENAKIS, M., 1995. Antimicrobial activity of mint essential oils. Journal of Agricultural and Food Chemistry, vol. 43, no. 9, pp. 2384-2388. http://dx.doi.org/10.1021/jf00057a013
    » http://dx.doi.org/10.1021/jf00057a013
  • SNOUSSI, M., NOUMI, E., TRABELSI, N., FLAMINI, G., PAPETTI, A. and DE FEO, V., 2015. Mentha spicata essential oil: chemical composition, antioxidant and antibacterial activities against planktonic and biofilm cultures of Vibrio spp. strains. Molecules, vol. 20, no. 8, pp. 14402-14424. http://dx.doi.org/10.3390/molecules200814402 PMid:26262604.
    » http://dx.doi.org/10.3390/molecules200814402
  • SULIEMAN, A.M.E., ABDELRAHMAN, S.E. and ABDEL RAHIM, A., 2011. Phytochemical analysis of local spearmint (Mentha spicata) leaves and detection of the antimicrobial activity of its oil. Journal of Microbiology Research, vol. 1, no. 1, pp. 1-4. http://dx.doi.org/10.5923/j.microbiology.20110101.01
    » http://dx.doi.org/10.5923/j.microbiology.20110101.01
  • SWAMY, M.K., AKHTAR, M.S. and SINNIAH, U.R., 2016. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: an updated review. Evidence-Based Complementary and Alternative Medicine, vol. 2016, pp. 3012462. http://dx.doi.org/10.1155/2016/3012462 PMid:28090211.
    » http://dx.doi.org/10.1155/2016/3012462
  • TOMI, K., KITAO, M., KONISHI, N., MURAKAMI, H., MATSUMURA, Y. and HAYASHI, T., 2016. Enantioselective GC-MS analysis of volatile components from rosemary (Rosmarinus officinalis L.) essential oils and hydrosols. Bioscience, Biotechnology, and Biochemistry, vol. 80, no. 5, pp. 840-847. http://dx.doi.org/10.1080/09168451.2016.1146066 PMid:26923429.
    » http://dx.doi.org/10.1080/09168451.2016.1146066
  • UGBABE, G.E., OKHALE, S.E., EGHAREVBA, H.O. and IBRAHIM, J.A., 2016. Foliar microscopy and GC-MS analysis of the volatile oil constituents of the leaf of Cymbopogon citratus (DC.) Stapt. (Poaceae/Graminae). International Journal of Basic and Applied Sciences, vol. 5, pp. 45-49.
  • WANNISSORN, B., JARIKASEM, S., SIRIWANGCHAI, T. and THUBTHIMTHED, S., 2005. Antibacterial properties of essential oils from Thai medicinal plants. Fitoterapia, vol. 76, no. 2, pp. 233-236. http://dx.doi.org/10.1016/j.fitote.2004.12.009 PMid:15752638.
    » http://dx.doi.org/10.1016/j.fitote.2004.12.009
  • WEINSTEIN, M.P. and LEWIS II, J.S., 2020. The clinical and laboratory standards institute subcommittee on antimicrobial susceptibility testing: background, organization, functions, and processes. Journal of Clinical Microbiology, vol. 58, no. 3, pp. e01864-e01819. http://dx.doi.org/10.1128/JCM.01864-19 PMid:31915289.
    » http://dx.doi.org/10.1128/JCM.01864-19
  • WIŃSKA, K., MĄCZKA, W., ŁYCZKO, J., GRABARCZYK, M., CZUBASZEK, A. and SZUMNY, A., 2019. Essential oils as antimicrobial agents: myth or real alternative? Molecules, vol. 24, no. 11, pp. 2130. http://dx.doi.org/10.3390/molecules24112130 PMid:31195752.
    » http://dx.doi.org/10.3390/molecules24112130
  • YAP, P.S.X., YIAP, B.C., PING, H.C. and LIM, S.H.E., 2014. Essential oils, a new horizon in combating bacterial antibiotic resistance. The Open Microbiology Journal, vol. 8, no. 1, pp. 6-14. http://dx.doi.org/10.2174/1874285801408010006 PMid:24627729.
    » http://dx.doi.org/10.2174/1874285801408010006

Publication Dates

  • Publication in this collection
    09 May 2022
  • Date of issue
    2024

History

  • Received
    21 Dec 2021
  • Accepted
    16 Mar 2022
Instituto Internacional de Ecologia R. Bento Carlos, 750, 13560-660 São Carlos SP - Brasil, Tel. e Fax: (55 16) 3362-5400 - São Carlos - SP - Brazil
E-mail: bjb@bjb.com.br