The assessment of good and bad bacteria in holy basil ( Ocimum sanctum ) leaves

Holy basil (Tulsi, Ocimum sanctum ) is considered to be a pious plant. It is used not only in many of the rituals of Hindus but is also known for its innumerable medicinal qualities. Holy basil leaves (HBLs) are often consumed raw thus it is pertinent to detect the bacteria on Tulsi leaves. This study was conducted to understand the diversity of the aerobically growing microbiome of edible holy basil leaves. A total of 112 samples of leaves were collected from households of six localities in and around Bareilly, India for detecting culturable bacteria growing aerobically. A sum of 579 bacterial isolates belonging to 106 different species was identified. Pantoea agglomerans isolates were detected in 43 samples followed by isolates

Submit a manuscript: https://www.tmrjournals.com/idrIntroduction Holy basil (Tulsi, Ocimum sanctum Linn), a member of the Lamiaceae family's Ocimum genus, and native to the Indian subcontinent grows throughout Southeast Asia and other parts of the world.It is widely used in Ayurvedic and folk medicine, often as a fresh leaf extract or herbal tea for a variety of ailments.It is considered sacred in Hinduism and has been described as the "Queen of plants" and the "mother medicine of nature" due to its perceived medicinal qualities [1].It is an integral culinary herb used in cooking especially pious foods like Prasad, Charnamrit and Panchamrit [2].Ocimum sanctum is said to be a valuable nutraceutical [3] due to its lethal effect on potential pathogens like Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa [4], and its ability to modulate the gut microbiota [5].Besides, holy basil is also claimed to diffuse stress [6,7], and exerts antioxidant [8], anti-inflammatory [9], hypoglycemic [7,10], hepato-protective [11], and anticancer [12,13] effects.It is shown to increase growth, disease resistance, and immune response in fish against bacterial and fungal infections [14,15].
Holy Basil (Ocimum sanctum), is a rich source of essential oils [16], and has potent antimicrobial activity against a wide range of pathogens [4,17].In ethnic medicine, Tulsi is used in chewing or just engulfing fresh leaves, extract from leaves, fresh or dried leaves in herbal teas or with other herbs to potentiate their medicinal value for the treatment of poisoning, stomach-ache, common colds, headaches, malaria, inflammation, and heart disease [18].Holy basil oil besides being a potent antimicrobial [17] is also valued for its expectorant, analgesic, anti-emetic, antipyretic; stress reducers and anti-asthmatic, hypoglycemic, hepatoprotective, hypotensive, hypolipidemic, and immunomodulatory activities [1].Due to the pious value of holy basil in Indian culture majority of Hindus and other people either take fresh leaves every morning or consume leaves as an integral ingredient of pious foods (prasads) and drinks (Charnamrit and panchamrit) offered to deities and worshippers [2].Though it has been known for a long that bacteria like Pseudomonas cichorii and P. virdiflava may cause bacterial leaf spot disease in holy basil [19,20], little is understood about bacteria present on edible holy basil leaves, which may be good (non-harming) or bad (pathogenic).This study was undertaken to detect aerobically growing bacteria present in holy basil leaves collected from houses of people who usually consume fresh leaves and use them in puja prasads.

Materials and methods
Collection of Ocimum sanctum (Rama Tulsi) leaves: Taking into the practice of consuming O. sanctum (Holy basil/ Tulasi) leaves (HBLs) in the morning, samples were collected between 7-8 am from September 2022 to May 2023.Mature fresh leaves (7-10) from plants of Tulsi, planted inside premises of households (leaves of which are usually consumed raw in different forms by the house owners) were picked up with sterile forceps from 3-5 twigs of each plant at the height of 1.5-2.5 feet.Leaves were transferred to a sterile screw-capped tube.Separate sterile pairs of forceps and collection tubes were used for the collection of Tulsi leaves from different household samples.The HBL samples were collected from five different urban localities (in Bareilly city), 1) ICAR-Indian Veterinary Research Institute, Izatnagar, 2) Mahanagar, Bareilly, 3) Suncity, Bareilly, 4) Rajendra Nagar, Bareilly, North City, Bareilly, and one rural area near Bhojipura, Bareilly, Uttar Pradesh, India.
Processing samples for isolation of bacteria: After bringing the samples to the laboratory within an hour of collection, in each sample tube, 10 mL of buffered peptone water (B.P.W., Difco, U.S.A.) containing 0.1% of tween-80 (Sigma, U.S.A.) was added.All tubes were swirled for a minute and incubated on a shaking platform at 37 °C for 6-8 h.Thereafter, from each of the sample tube, a loop full of contents was streaked onto blood agar (containing 5% defibrinated sheep blood) plates and incubated for 18-24 h at 37 °C.Isolated colonies of different types (5)(6)(7)(8)(9)(10) were picked up and re-streaked on fresh blood agar plates for purity test.Isolates showing pure growth were characterised using growth, morphological, staining and biochemical characteristics to identify the bacteria [21].Impure isolates were separated and characterized similarly.All pure cultures were maintained on nutrient agar slants at 4 °C, and in 25% glycerol broth [22] at -20 °C throughout the study.Those isolates that could not be confirmed with conventional bacteriological methods were subjected to Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification using the MALDI Biotyper® Sirius system.
All the isolates of bacteria from HBLs were also tested for their herbal antimicrobial susceptibility again ajowan (Trachyspermum ammi) oil (AO), carvacrol (Sigma, U.S.A.), cinnamaldehyde (CNH) (Sigma, U.S.A.), Cinnamon (Cinnamomum verum) oil (CO), citral (Sigma, U.S.A.), holy basil (Ocimum sanctum) oil (HBO), lemongrass (Cymbopogon citratus) oil (LGO), methanolic extract from leaves of gooseberry (Phyllanthus embalica), Polyalthia longifolia seed oil, sandalwood (Santalum album) oil (SWO), and thyme (Thymus vulgaris) oil (TO).Except for methanolic extract from leaves of gooseberry (Phyllanthus embalica), all herbal oils were procured with > 99.5% purity from Naga Fragrance Ltd, Dimapur, India.Methanolic extract of gooseberry leaves was prepared from dried leaves' powder by soaking 20 g of leaf powder in 100 mL of methanol at 20 °C for 24 h at a shaking platform and then collecting supernatant, dried after filter sterilization at 50 °C for 12 h and then dried contents were weighed and again dissolved in pure ethanol to make 20 mg/ mL solution.The discs loaded with 1mg of herbal compound/ oil were prepared and stored in airtight vials at 4 °C till used for susceptibility testing [24].The growth inhibition zones around herbal discs were measured in mm, and the isolate with inhibition zone diameter ≥ 8 mm indicated susceptibility of the isolate else listed as resistant.The isolates resistant to three or more herbals were counted as multiple herbal antimicrobial-resistant (MHDR).
Statistical analysis: Data on bacterial identification and susceptibility of isolates were entered in an Excel sheet, and were analysed using Chi-square and Odds ratio to conclude any relationship.

Results
A total of 579 bacterial isolates belonging to 106 different species were identified from 112 samples of HBLs in the study.The most commonly isolated bacteria (from ≥ 11 samples) were Pantoea agglomerans detected in 43 samples followed by Virgibacillus pantothenticus (21), Bacillus coagulans (18), Bacillus cereus (17), Geobacillus stearothermophilus (16), Klebsiella pneumoniae (13), Citrobacter freundii (12), Lysinibacillus sphaericus (12), Acinetobacter calcoaceticus (11), Escherichia coli (11), and Xenorhabdus bovienii (11).Bacteria belonging to 44 species were isolated from only one sample each (Table 1).A total of 161 isolates of putatively good bacteria belonging to eight genera (Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, Photobacter, Siccibacter, Virgibacillus, Xenoirhabdus) having one or other beneficial use for health, industry or agriculture were isolated from 68 (60.71%) samples of HBL while 418 isolates of potentially pathogenic bacteria for human and animals (belonging to 34 genera) were detected in 108 (96.43%) of the HBL samples.There were only four samples (three from Mahanagar and one from Bhojipura) which had no potential pathogenic bacteria detected in this study.Of the 30, 29, 20, 5, 5, and 23 HBL samples collected from a rural area of Bhojipura and an urban campus of Indian Veterinary Research Institute (IVRI) Izatnagar, Mahanagar, North City, Rajendra Nagar and Sun City, 26, 9, 16, 5, 4, and 8 samples, respectively had good bacteria.The analysis indicated that HBL leaves collected from the ICAR-IVRI campus had the least probability of harbouring good bacteria significantly (P < 0.04) lower than samples from Bhojpura, Mahanagar, North City and Rajendra Nagar; the next poor source for good bacteria were the samples from the Suncity, significantly poorer than samples from Bhojpura, Mahanagar, and North City.The best source of good bacteria was the holy basil leaves collected from rural areas of Bhojipura and urban households in North City, and Mahanagar.
Antimicrobial resistance in non-pathogenic or good bacteria was significantly more common (P < 0.01) for ajowan oil, but they were more often (P < 0.05) sensitive to holy basil oil, lemongrass oil, citral, P. embalica leaf extract, P. longifolia seed oil, nitrofurantoin, ciprofloxacin, chloramphenicol, colistin and minocycline than isolates Submit a manuscript: https://www.tmrjournals.com/idr Bacillus badius Bacillus subtilis  Acinetobacter ursingii Streptococcus mitis 1 (1)  Burkholderia cepacia Enterococcus solitarius Pluralibacter gergoviae of potentially pathogenic bacteria.For other antimicrobials, there was no significant difference (P > 0.05) between the susceptibility of good and potentially pathogenic bacteria, but good bacteria were often more susceptible than that were potentially pathogenic, except for the cinnamaldehyde situation reversed.However, there was a wide variation in antimicrobial susceptibility for herbal and conventional antibiotics used for treating clinical infection among strains of different species (Table 3 and 4).
Discussion Submit a manuscript: https://www.tmrjournals.com/idrDetection of a variety of bacteria on holy basil leaves is neither unnatural nor alarming because bacteria are ubiquitous in nature and present everywhere in all biotic and abiotic environments.A similar profile of bacteria is reported on vegetables, fruits, plant products and even leaves of Azadirhacta indica, a plant revered for its antibacterial potential, like holy basil [25][26][27].However, the prominence of potentially pathogenic bacteria over good bacteria is of concern and might be associated with pollution [28] especially the biotic pollution indicated by a high number of samples of HBLs devoid of good bacteria from ICAR-Indian Veterinary Research Institute, Izatnagar, where studies are conducted on bacteria and lot many sick animals and humans visit the veterinary and medical hospitals in the campus.Most of the bacteria isolated from HBL samples were susceptible to cinnamaldehyde and carvacrol, the active antimicrobial compounds in cinnamon oil and thyme/ ajowan oil, respectively.These two compounds have often been reported as the best antibacterial on both environmental and clinical isolates of bacteria [29,30].Multiple drug resistance was common among both, good (44.72%) and potentially pathogenic bacteria isolates (67.92%) from HBLs in the study and the occurrence of MDR is almost similar as observed in bacteria present on A. indica leaves in a similar locality [25] indicating the environmental circulation of MDR bacteria.However, it is important to detect carbapenem-resistant bacteria on five of the HBL samples (three from rural areas and two from urban areas) because they are considered to be superbugs if they also possess pathogenic potential [31].
A total of 47.15% bacterial isolates from HBLs were resistant to holy basil oil (HBO, oil extracted from holy basil leaves).The HBO is considered a potent antibacterial.However, the presence of HBO susceptible bacteria on HBLs might be either due to recent contamination of HBLs or low concentration of HBO at the lodging site of bacteria on HBLs or just isolation of bacteria living in stationary phase on HBLs.
Among the most prevalent bacteria on HBLs, P. agglomerans was detected in 38.39% of samples in the study.It is a ubiquitous bacterium on plants, often beneficial to plants exerting antifungal activity, producing phytohormones and fixing nitrogen, and protects sugar cane (Saccharum officinale) from Lead-scald disease caused by Xanthomonas albilineans [32].However, it is an opportunistic pathogen in humans and animals [33] thus consumption of contaminated or infested HBLs may be risky for the health of consumers.
The next common bacterium detected in 18.75% of HBL samples was Virgibacillus pantothenticus, rarely reported as a cause of illness in humans.However, it has commonly been reported in food, water, soil and in antacid preparations and is considered a friendly bacterium [34].Recently, it is also been detected in the urine of apparently healthy cattle [31].
Another commonly isolated bacterium from HBLs was Bacillus coagulans.It is a type of "good" bacterium [35]; it has a modulating effect on gut and vaginal microbiota and may be beneficial for irritable bowel syndrome (IBS).It was present in 16.17% of HBL samples.However, the majority of the isolates had MDR which is a matter of concern as 'R' factors (genes responsible for the transmission of antibiotic resistance) are transferable [30].
Detection of Bacillus cereus on 15.18% of HBL samples may be of public health concern because HBLs are often added raw in many of the pious foods/prasads.Strains of B. cereus have often been incriminated as a cause of food poisoning [2].Bacillus cereus is also reported as a cause of serious and potentially fatal non-gastrointestinal tract infections.It is of common occurrence in decaying organic matter, fresh and marine waters, vegetables and fomites, and in the intestinal tract of invertebrates, and their excreta may contaminate soil and food products [36].Besides, several other bacilli were also detected on HBLs including B. circulans (5), B. licheniformis (5), B. subtilis (4), B. lentus (2), B. thurigiensis (2), B. badius (1), and B. firmus (1).Though B. circulans is considered a soil bacterium good for plant growth, it has been associated with fatal sepsis, septicaemia, meningitis, endocarditis, endophtalmitis, wound and joint infections [37] thus its detection on HBLs may be a health hazard.
Another common bacillus on HBL, B. licheniformis, widely distributed in environments including plants [25], is considered a friendly bacterium having probiotic potential [38].Bacillus subtilis isolated in four HBL samples, is also a good bacterium used as a probiotic [39], but may be equally dangerous if the strains are toxigenic, and has been incriminated in many food-borne disease outbreaks [40].Similarly, B. lentus is also a very useful bacterium assisting in digestion through the production of many enzymes like α-amylase, β-glucanase, hemicellulase, xylanase, protease (subtilisin) and alkaline phosphatase.Serine proteases from B. lentus are reported to kill even prions [41].In the same row, good bacteria, B. thurigiensis and B. firmus, were also detected on HBLs.They are widely used as biocontrol weapons against agricultural pests, and nematodes [42], respectively.Strains of B. badius isolated on a few HBLs are also classified as safe for humans and are a source of several useful bio-molecules [43].
Strains of Geobacillus stearothermophilus detected in 14.29% of HBL samples have niches in a variety of environments including hot springs, frozen soils, food manufacturing plants, and dairy plants due to flat sour spoilage of low-acid foods [44].
Strains of Klebsiella pneumoniae ssp.pneumoniae were detected in 11.61% of HBL samples.This is a nasty pathogen causing pneumonia, and gastrointestinal infections, and is one of the most common causes of nosocomially acquired multiple drug-resistant (MDR) and total drug-resistant (TDR) systemic illnesses [45].It is also a commensally occurring bacterium even in vegetables, healthy humans and animals and is excreted in urine and faeces [31,46].Besides, K. oxytoca, K. aerognese and K. aeromobilis were also detected in 3, 1 and 1HBL leave samples, respectively.Strains of K. oxytoca are known to cause serious and lethal septicaemia and respiratory tract infections [47], and also colonize other plants including A. indica leaves [25,46].The strains of K. aerogenes are often the cause of nosocomial infections [48] and strains of K. aeromobilis are linked with inflammatory bowel disease (IBD), an emerging problem, and also with other intestinal infections [49].The detection of klebsiellae on HBL leaves becomes important as the majority of the isolates (17 of 25) had MDR, and they were considered important pathogens.
A total of 10.71% of HBL samples harboured Citrobacter freundii strains.Besides, one sample had two strains of C. amalonaticus.Citrobacters are ubiquitous types of bacteria found in water, soil, food, and intestinal tracts of humans and animals.The majority (14/17) of the isolates were MDR type and three produced extended-spectrum β-lactamses (ESBL).Earlier, Citrobacter strains were considered commensals or contaminants, now they are known to cause a wide spectrum of infections and MDR-nosocomial infections of the urinary tract, liver, biliary tract, peritoneum, intestines, bones, respiratory tract, heart, wounds, meninges and even the bloodstream [50].
Strains of Lysinibacillus sphaericus were detected in 10.71% of the HBL samples in the study.It is a natural inhabitant of soil and can be considered a friendly bacteria as some of the strains are highly toxic to Culex sp, Anopheles sp, Psorophora sp, and to some extent, to Aedes sp.mosquito larvae [51].However, the detection of carbapenem-resistant L. sphaericus strain in the study might be of concern due to the possibility of the spread of carbapenem resistance to other environmental bacteria [30].
Acinetobacter strains isolated from 16.96% of HBL samples (A.calcoaceticus 11, A. lwoffii 4, A. schindleri 1, and A. ursingii 1) in the study are common inhabitants in patients with or without causing infections.They are considered important nosocomial pathogens associated with lung (pneumonia), blood (septicaemia), wound, or urinary tract infections (UTI).They are an important member of the ESKAPE group of members causing infections both in humans and animals [17,25], especially strains of A. calcoaceticus-baumannii complex [52], those were the most common Acinetobacter strains in HBL samples.
Isolation of Escherichia coli from 9.82% of holy basil leaves samples is alarming.Strains of E. coli are ubiquitous opportunistic pathogens causing highly drug-resistant infections in humans, animals and birds Submit a manuscript: https://www.tmrjournals.com/idr[53].It has also been reported as equally prevalent (10%) on neem (Azadirachta indica) leaves in the Bareilly region showing its versatility to adapt to different hosts and niches [25].Besides E. coli, strains of three more species of Escherichia (E.vulneris 6, E. fergusonii 1, and E. hermanii, 1) were also detected on HBLs.Six of the HBL samples had seven E. vulneris strains, five of which were MDR-type and resistant to holy basil essential oil's antimicrobial action.Isolation of E. vulneris is an important indication of the risk associated with unclean HBLs because, unlike E. coli, E. vulneris is often considered more pathogenic to humans causing persistent diarrhoea, sepsis due to their ability to produce haemolysins and heat-labile enterotoxins, suppurative lymphadenopathy, meningitis, peritonitis, wound infections and UTI in humans, and abortions in cows, septicemic deaths in birds, Himalayan black bears and Gaur ungulates, diarrhoea in birds, and enteritis in tigers [54].Birds are considered the potential disseminator of MDR strains of E. vulneris, but in another study in the same region E. vulneris were rare on A. indica leaves [25] despite the fact that birds may be perching more on A. indica tree than on smaller holy basil shrubs.Similar to E. coli and E. vulneris, E. hermanii [55] and E. fergusonii [56] strains may also cause serious infections, leading to abortions, septicemic death, diarrhoea, endometritis, enteritis, mandibular abscesses, prostatitis, and urinary tract infection in humans, animals and bird [54].Thus, the presence of strains of Escherichia species on 14.29% of HBLs, used without any curing, may be a potential health hazard.
Bacillus megaterium, a plant growth-promoting bacterium reported to enhance the growth of seedlings through complementation of plant cytokinin receptors [57] was detected on 8.04% of holy basil leaves' samples indicating the possible utility of holy basil extract as a source of this useful bacterium for agriculture.
Strains of Hafnia alvei were present on 8.04% of HBL samples.It is an opportunistic pathogen and has been reported as an emerging cause of nosocomial infections in humans.It is a natural inhabitant in the environment (soil, water, and sewage) and gastrointestinal tract of animals but has also been reported in outbreaks of disease in a variety of animal species such as cows, goats, chickens, mares, ducks, snails, fish and bees [58].Recently H. alvei has been reported as one of the most common bacteria in the urine of herbivores [31] and on leaves of A. indica [25].
Strains of Paenibacillus alvei and P. larvae are the nasty causes of European foulbrood disease and American foulbrood disease of honey bees, respectively leading to considerable economic losses to honey bee breeders [59].Isolation of P. alvei and P. larvae strains each from 8.04% of HBL samples needs to be further investigated to assess their role in any illness in honey bees in the Bareilly region.Paenialvin A to D are four antimicrobial peptides produced by some of the P. alvei strains that inhibit methicillin-resistant Staphalococcus aureus (MRSA), Bacillus subtilis, Loktanella hongkongensis, E. coli, and Pseudomonas aeruginosa, and kills even potential foodborne bacteria like salmonellae but cytotoxic effect on human cell lines limits their utility as therapeutic agent [60].However, many of the P. alvei strains remained on the list of potentially harmful bacteria.Besides, one sample of each of the HBLs was positive for P. lentimorbus and P. papillae.All the isolates were susceptible to most of the antimicrobials used in the study.The former is reported as a growth stimulant for tobacco plants, and protects plants from viral infections [61], and later is a known pesticide for several harmful pests in agriculture [62].
Paenibacillus macerans often found in soil and plants are considered useful bacteria capable of phosphorus solubilisation and have been used as a bio-fertilizer [62].Many of the P. macerans strains kill plant pathogens and have also been associated with recurrent wound infection in humans [63].
Raoultella terrigena, earlier known as Klebsiella terrigena, detected in 7.14% of HBL samples in the study, is an environmental bacterium that recently emerged as an MDR pathogen causing numerous ailments in humans and animals [54].It has also been detected in 6.37% of A. indica plants [25].Detection of R. terrigena strains on HBL is of public health concern due to their potential pathogenicity in humans.
Enterococcus faecium, E. faecalis and E. solitaries were isolated from 6, 5, and one sample, respectively, and a sample had both E. faecalis and E. faecium.From three samples carbapenem-resistant E. faecium and from one E. faecalis were detected.Of the three enterococci, E. faecium is a serious nosocomial pathogen often showing resistance to carbapenems [64] as observed in the present study.Besides being resistant to most of the drugs, both E. faecalis and E. faecium are good colonizers in intestinal tracts of humans, birds and animals and are potentially pathogenic causing a variety of infections viz., endocarditis, urinary tract infections, prostatitis, intra-abdominal infection, cellulitis, and wound infections, and bacteraemia [65,66].A similar level of occurrence of enterococci has been reported on A. indica leaves in a similar region to the present study [25]; still higher level of presence was detected in urine samples of herbivore animals [31].
Though Xenorhabdus bovienii has been reported from clinical and environmental samples, it usually colonizes in the intestine of the infective stage of the entomopathogenic nematodes [67].In animals, X. Bovienii strains have been reported to cause abortion in bovines, abscess septicaemia and diarrhoea [54].In the present study, X. bovienii isolation was made from 9 HBL (8.04%) samples while less than 1% of A. indica leaves' samples had this bacterium in the Bareilly region [25].Besides, one isolate each of Photorhabdus luminescens (earlier known as X. luminescens), and X. nematophilus was identified from two different samples of HBLs.Strains of both of these species are reported as lethal pathogens of insects and a potential pesticide [67].
Of the 112 HBL samples examined only six had Bacillus mycoides.Strains of B. mycoides are present in a wide variety of environments, including soil and water [68].Recently, they were detected as one of the most common bacteria in the urine of herbivores [31], but rare on leaves of A. indica [25].
Strains of Lellitio amnigenus (L.amnigena, earlier known as Enterobacter amnigenus) were isolated from six HBL samples, and L. nimipressuralis from five of the HBL samples.Lellitio strains are not only considered human pathogens but also affect plants [69].Isolation of L. amnigenus has been reported from cases of UTI in humans [70] thus, the presence of Lellittio strains on HBLs (consumed raw) may be of public health concern.
Isolates of Pseudomonas aeruginosa were identified from six of the HBL samples.It is an important MDR-type ESKAPE pathogen of nosocomial importance causing a wide spectrum of infections in humans and animals [71], thus, their detection on HBL is of concern.Though P. aeruginosa is one of the ubiquitous bacterium surviving and growing under wide variations of environments, it was reported to be a less common inhabitant (< 2%) on A. indica leaves [25], and has rarely been isolated from healthy humans and animals.
A total of 15 HBL samples had one or other aeromonad (Aeromonas bestiarum, 2; A. eucranophila, 1; A. hydrophila, 1; A. media, 3; A. popoffii, 1; A. salmonicida ssp.salmonicida, 3; A. schubertii, 2; A. trota, 5).Strains of A. bestiarum and A. caviae have also been reported on A. indica leaves in similar locations as of this study and might be associated with the environmental spread of the potential pathogen [25].On plants, aeromonads considered the beneficial bacteria protecting from dehydration, are usually associated with disease causation in humans, fish and animals [72].Though many herbs are reported to kill aeromonads including cinnamon, garlic, lemongrass and holy basil [73], > 45% of the isolates in the present study were resistant to holy basil oil, citral, and sandalwood oil thus, the presence of aeromonads on HBLs is of public health concern.
Brevibacillus brevis strains were detected in five HBL samples.They are known to produce gramicidin, an antimicrobial peptide, a potential antifungal and a plant growth promoter for cotton crops [74].However, Brevibacillus species are rarely been reported as a cause of disease in immunocompromised hosts, intravenous drug users, victims of burns and physical trauma, dialysis patients, and patients having orthopaedic and neurosurgical surgery procedures [75], thus are not totally good type of bacteria.
Strains of Erwinia often reported as plant pathogens causing Submit a manuscript: https://www.tmrjournals.com/idrbacterial rot in fruits and vegetables were identified in 14 HBL samples.A total of 18 isolates in the study belonged to E. aphidicola, 7; E. mallotivora, 4; E. amylovora, 2; E. ananas, 2; E. nigrifluence, 1, and E. uredovora, 2. Ervinia strains are recently reported to cause a wide spectrum of clinical infections in humans, animals and birds [76] and have also been isolated from A. indica leaves [25] in the Bareilly region.
Strains of Kluyvera genus (K.cryocrescens 2, K. ascorbata 1, and K. intermedia 1) were detected on a few samples of HBLs but all four isolates had extensive drug resistance and one isolate also produced ESBL.They are known as progenitors of extended-spectrum β-lactamases and are isolated from clinical samples from repeat breeding, metritis, septicaemia and enteritis cases in animals, and are prevalent in the environment [54].Therefore, Kluyvera strains are considered potentially dangerous pathogens if present in foods due to their ability to cause persistent and chronic infections.
Another common group of bacteria on HBLs was Pectobacterium spp.(P.cyperipedii 5, P. carotovorum 4, P. chrysanthemi 4, P. rhapontici 4, and P. cacticida, 2) were detected in 16 samples (14.29%).Pectobacterium strains producing pectolytic enzymes are serious pathogens of vegetables, fruits and plants causing soft rot [77], and have also been reported on A. indica leaves [25].Pectobacterium strains are not only the cause of plant pathogenesis but have also been reported in association with abortion, diarrhoea, septicemic death, pneumonic lungs, mastitis, and UTI cases in humans and animals [76].Therefore, the presence of Pectobacterium strains on HBLs may be of public health concern specifically due to the raw consumption of HBLs [2].
Serratia strains, often considered environmental bacteria, were detected in 16 HBL samples (14.29%) in the study.They are also known to cause several diseases and nosocomial infections in animals and humans due to the production of various virulence factors and extended-spectrum β-lactamases [78].Besides, Serratia strains can cause infection in plants and may interfere with seed germination [79].Thus, the isolation of Serratia strains from 16 HBL samples is of immense significance.
Staphylococcus strains (S. aureus 4, S. epidermidis 3, S. capitis ssp.capitis 1, S. kloosii 1, S. lentus 1, and S. (Mammaliicoccus) sciuri 1) detected in 11 HBL samples (~10%) is of public health significance.Staphylococci are normal inhabitants on human and animal skins but less common on plants.They cause skin infections, bacteraemia, bone infections, endocarditis, pneumonia, and toxic shock syndrome in susceptible hosts [80].Strains of S. aureus, one of the most common staphylococci detected in HBL samples, are a major cause of food poisoning through the production of comparatively heat-stable enterotoxins [80].Thus, the use of raw and fresh leaves of holy basil in prasad [2] may be dangerous if carrying toxigenic staphylococci.Though there is a recent report on the probiotic potential of M. sciuri, it is still considered a pathogen due to hosting several virulence factors [81].
Isolates of Cronobacter sakazakii (earlier known as Enterobacter sakazakii) were identified from three samples, and C. malonaticus from one HBL sample.These bacteria are known as opportunistic food-borne pathogens affecting a wide range of hosts [82].It is one of the important causes of infant and neonatal infections and is often transmitted through contaminated foods [82].Thus, its presence in HBLs is of importance as these are often consumed raw and untreated.Multiple drug-resistant Alcaligenes denitrificans isolated from four HBL samples are of public health importance due to their high level of drug resistance and ability to form persistent biofilms [83].Isolation of an ESBL-producing MDR strain of Achromobacter xyloxidans, a nasty nosocomial infective agent, in a sample of HBL collected at Suncity Vistaar, Bareilly, is of concern due to its emergence as difficult-to-treat infection [83].A strain of MDR Burkholderia cepacia was detected in a sample of HBL collected at the Institute campus is also an important pathogen causing highly drug-resistant biofilms and is difficult to eliminate contaminants detected even in disinfectant solutions [84,85].Isolation of Buttiauxella agrestis from an HBL sample seems to be of concern due to the nosocomial potential of this pathogen [86], however, the isolate in the present study was neither ESBL-producer nor MDR type.
Two samples of HBLs had Cedecia lapagei, an opportunistic pathogen causing soft tissue infections, pneumonia, urinary tract infections, wound infections, peritonitis, osteomyelitis, bacteremia, sepsis, and enterocolitis in humans, and mastitis in cow [54].One of the isolates was the MDR-type, thus its presence on HBLs is important for the HBL consumers' health.
Isolation of holy basil essential oil susceptible and non-MDR Aerococcus sanguinicola, an emerging pathogen causing clinical infections [87], from a single sample of HBL from the Mahanagar area, may be important.Aerococcus strains have also been reported on A. indica leaves in Bareilly at much higher positivity [25]; the lower occurrence in HBL may be due to the susceptibility of aerococci to holy basil essential oil.
MDR strains of Enterobacter species (E.cloacae subsp.dissolvens 4, E. intermedius 1 and E. taylorae 1) were detected in six samples of HBLs from four out of the six sampling sites, of these three were also positive for ESBL production.Strains of Enterobacter species are common in the environment [76] and may cause diseases in plants [88], reside on healthy A. indica leaves [25], and also in humans and animals [76].Enterobacter species strains are usually associated with nosocomial infections, but may also be community-acquired infections causing UTI, respiratory infections, soft tissue infections, osteomyelitis, and endocarditis, and becoming more important due to their MDR nature [89].
A non-MDR strain of Ewingella americana was isolated from an HBL sample from a household in Suncity.Its isolation is important as this bacterium has been identified as an emerging pathogen causing serious and sometimes lethal infections like peritonitis, conjunctivitis, bacteremia, pneumonia, meningitis, and wound infections [90].
From a sample from a rural household of Bhojipura a Flavimonas oryzihabitans strain was isolated.Its importance can be signified by the fact that it may cause severe bacteraemia in humans [91].
Isolation of Moraxella osloensis, an emerging pathogen, causing several types of nosocomial infections and bacteraemia [92] is important being present on a plant that is considered holy and leaves are consumed raw.
Pluralibacter gregoviae, earlier known as Enterobacter gregoviae, is a potential pathogen known for its high-level drug resistance [93], its detection on HBLs is of concern as the isolates were resistant to the most common antibiotics like nitrofurantoin, tetracycline, chloramphenicol and minocycline.
Proteus mirabilis is a very common bacterium in soil and water environments sometimes associated with catheter-associated urinary tract infections (CAUTIs) and a variety of other infections due to its highly resistant and persistent bio-films in the environment [94].It was detected from one sample of HBLs indicating that holy basil may be dealing with this pathogen efficiently but the isolate recovered was resistant to holy basil essential oil and three of the common antibiotics (colistin, ceftriaxone and nitrofurantoin) tested.A similar type of P. mirabilis has also been reported on A. indica leaves in the Bareilly region [25].
Isolates of Providencia rustgianii and P. rettgeri were isolated from two and one HBL samples, respectively.All isolates had MDR, and the isolate of P. rettgeri was also resistant to meropenem qualifying it to be a superbug.Strains of Providencia sp., opportunistic bacteria, have been reported to cause mastitis in cows and wound infections in pigs [54].
Isolation of Salmonella enterica ssp.indica strains from two HBL samples in this study seem to be novel and alarming findings as salmonellae are rarely reported on plants as they mostly affect warm-blooded animals.Though, salmonellae are a common cause of infections in humans and animals [95], rarely affect plants [26].The presence of Salmonella strains in any of the foods is always considered a big risk factor due to its high pathogenic potential [95].
The identification of Siccibacter turicensis from HBLs is important due to its cellulose-degrading potential [96].It is frequently reported in scats of kangaroos and is supposed to help the animal in the Submit a manuscript: https://www.tmrjournals.com/idrdigestion of high-fibre diets [96].Strains of S. turicensis are rarely reported from other places, thus, it might be an important finding indicating a new niche for the bacteria in HBLs.
Strains of Streptococcus milleri, S. mitis and S. pyogenes were isolated from one HBL sample each and all four isolates had MDR.Strains of S. milleri are recognised as important emerging pulmonary pathogens associated with pleural empyema or lung abscess [97].On the other hand, S. pyogenes, a group 'A' pathogenic streptococcus, is known to cause a variety of ailments in humans and animals, causing millions of cases of sore throats the world over.The isolate from HBLs was resistant to all the herbal antimicrobials tested in the study and had MDR for antibiotics too.Another streptococcus isolated from HBLs, S. mitis, is a common inhabitant of the oral cavity.It rarely causes any disease but sometimes it has been identified as the cause of serious infections of heart, and infective endocarditis, especially in immunocompromised and cancer patients [98].
The detection of highly drug-resistant and potentially pathogenic bacteria on HBLs may be a serious health risk for the consumers of raw HBLs.Thus, it may be recommended that HBLs should be consumed after proper disinfection treatment(s) like putting leaves in hot teas (hot water may kill harmful bacteria) or washing with chlorinated water or by other means as recommended for decontamination of sprouts [99].
Detection of multiple antibiotic-resistances (MDR) in 61.49% of isolates and multiple herbal drug-resistances (MHDR) in 79.1% of bacteria isolated from HBLs is alarming and reflects the risk associated with the consumption of HBLs.However, similar levels of MDR and MHDR are common in bacteria isolated from clinical, environmental and food samples in the region of the present study [54,76,80].The study indicated that HBLs though considered pious and a source of antimicrobial HBO, are not spared of the carriage of drug-resistant microbes and get equally infested as other plants in the region [25].

Conclusion
The study revealed that holy basil plant leaves may harbour both good and bad bacteria; however, harmful bacteria dominated the spectrum of aerobic culturomes of HBLs.Thus, it should not be considered that the piousness of the plant is a surety for the biological safety of the plant for consumers.Detection of superbugs and a variety of zoonotically important bacteria on holy basil leaves suggests that the consumption of unclean and raw HBLs may be unsafe for consumers.

Table 2 Antimicrobial drug resistance in bacteria isolated from holy basil leaves collected from different sources Antimicrobial
tested Number of isolates resistant to antimicrobials isolated from holy basil leaves' sample collected from different areas samples collected (number of bacterial isolates)

Table 3 Herbal antimicrobial drug resistance among bacteria isolated from holy basil leaves
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Table 4 Antimicrobial drug resistance among bacteria isolated from holy basil leaves (Continuted)
1