1aResearch Center for Animal Production, CRA PCM, Monterotondo (RM), Italy
1bResearch Unit for the Extensive Animal Husbandry, CRA ZOE, Muro Lucano (PZ), Italy
2Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy
3Department of Environmental Biology, University of Rome Sapienza, Rome, Italy
Keywords: Wound-Contaminating-Bacteria; Antimicrobial Activity; Neem oil; Goat
A major concern emerging in recent years in the field of bacterial infections, which stimulate the development of innovative molecules with antimicrobial activity, is represented by the problem of multiple antibiotic resistance, i.e. onset and rapid growing global dissemination of pathogens that have acquired resistance to multiple antibacterial drugs in use, capable of supporting common infections, potentially fatal for animals and humans. According to World Health Organization (WHO, 2002-2005) [2] medicinal plants would be the best source to obtain a variety of drugs. In fact, many plants produce secondary metabolites which act towards wound-contaminating-bacteria and parasites [3,4]. Plants are a good source of antioxidants and also cure various disorders associated with wound inflammation [5]. Identification and studies on active compounds from plants are now of major interest to the scientific community [6]. These include compounds such as diterpenoid from raw plant material which showed antibacterial activity towards Streptococcus pyogenes and some fungi. In addition, tannins, found in a number of plants used in wound healing were also found to have antibacterial activity [7].
Nowadays, Neem (Azadirachta indica A. Juss) is considered an effective source of environmentally-powerful natural pesticide and considered to be one of the most promising pesticides. It is believed to be one of the trees of the 21st century for its great potential in pest management, environmental protection and medicine [8]. Among the many products obtained from the seeds, neem oil is the most commercially relevant (www.organicneem. com/why_parker_neem.html). More than four hundred substances were identified in the neem oil. The most biologically active are the nortriterpenoid limonoid. In particular, the azadirachtin A is the compound best known and studied for its properties that allows its use in agriculture as a natural insecticide, acaricide and nematocide, but it should not forget the salannin and nimbin, among other major limonoids. The insecticidal properties of neem oil are closely related to the larvicide, acaricide, nematicide, and repellent effects.
The antimicrobial activity of neem oil has been investigated [9,10] . In addition, it has been recently demonstrated the neem oil antibacterial activity towards several food spoilage bacteria [11] and strains of enteropathogenic Escherichia coli [12]. This study aimed at evaluating the neem oil antibacterial activity towards bacteria isolated from animal wounds resulting from ectoparasites sting or bite.
Mesophilic and thermophilic bacteria were counted on Dextrose Tryptone Agar (Oxoid, Garbagnate Milanese (MI), Italy), after incubation at 32°C for 72 hours and 55°C for 48 hours, respectively.
Coliforms with E. coli distinction were counted on Chromogenic Compact Dry and Chromogenic Compact Dry with Escherichia coli distinction (PBI, Milano, Italy) after incubation at 35°C for 20 hours [16, 17].
Baird Parker Agar (Acumedia, Neogen, lansing MI, USA) was used as selective medium for the isolation and presumptive identification of catalase-positive staphylococci. It contains 7.5% sodium chloride and thus selects for those bacteria which can tolerate high salt concentration.
Mannitol Salt Agar is both a selective and differential medium used in the isolation and distinction of S. aureus and S. epidermidis.
Catalase-negative Enterococcus spp. and Streptococcus spp. were isolated on Columbia Blood Agar Base, Streptococcus Selective Supplement and Defibrinated Horse Blood (OXOID, Basingstoke, UK) incubated plates at 37°C, either aerobically or anaerobically and examined after 18 to 24 hours incubation.
Pseudomonas Isolation Agar (Acumedia, Neogen Corporation, lansing, MI, USA) was used for the isolation of Pseudomonas aeruginosa and other Pseudomonas spp. under aerobic incubation at 35 ± 2°C and examined for growth after 18-48 hours. The medium includes Irgasan, a broad spectrum antibiotic not active towards Pseudomonas spp. This medium is selective and formulated to enhance formation of blue or blue-green pyocyanin pigment by Pseudomonas aeruginosa. The pigment diffuses into the medium surrounding growth.
Escherichia coli, American Type Culture Collection (ATCC)® 51813™, S. aureus subsp. aureus ATCC® 6538™, E. faecalis ATCC® 7088TM, and P. aeruginosa ATCC 27853 were used as reference strains.
To prepare working cultures, stock cultures were standardized through two successive 24 h growth cycles in Brain Heart Infusion broth (Difco, Buccinasco, MI, Italy) at 20° C without agitation.
The reference strains were grown on media and at the growth conditions as reported on products sheets.
Bacterial suspensions (100 μL), standardized by adjusting the optical density to OD 1 at 600 nm (Shimadzu UV-120-01 spectrophotometer, Shimadzu, Cinisello Balsamo, MI, Italy), were used to inoculate by flooding the surface of suitable agar media for each microorganism considered at their proper growth conditions. Excess liquid was air-dried under a sterile hood and the impregnated discs were applied at equidistant points on top of the agar medium. The plates were done in triplicates for each organism and the experiment was performed twice. Plates were checked after incubation at 35°C and 42°C for 48 h. Antimicrobial activity was measured as diameter (mm) of the inhibition zone around the disc. The results were recorded as means ± SD of the duplicate experiment.
The antibacterial activity of neem oil towards the bacteria was also evaluated in microdilution assays using conventional sterile polystyrene microplates. Each well of the microplate was filled with 100 μL of sterile suitable liquid media for each microorganism considered, 50 μL of inoculums (see Table 4) and amounts of extract at lower concentrations (1:10 to 1:100,000) were added. Control treatment without neem oil was used in the experiment. The microplates were incubated at 37°C for 24 h. Bacterial growth was determined by OD 0.02 reading at 630 nm/10 mm path length with an ELISA microplate reader (Dynatech ML-3000, Pina de Ebro, España). Bacterial cell concentration was transformed to cells/mL using the reference curve equation. The reference curve was constructed by diluting 1:100 each bacterial species. Counting the number of bacterial cells of an aliquot of this dilution was done using a Neubauer chamber (Celeromics, Vedano al Lambro, MI, Italy). Finally, cell concentrations were transformed to a percentage of bacterial inhibition. The percentage of bacterial growth reduction (GR%) was estimated using as reference the control treatment (without neem oil) as:
GR% = C – T/C x 100
Bacteria |
Growth media and conditions |
CFU / mL* |
Mesophilic |
DTA 32° C 72 h |
6 x 107 |
Thermophilic |
DTA 55° C 48 h |
2,3 x 104 |
Coliform |
CCD 35° C 20 h |
5,3 x 107 |
E. coli |
CCD EC 35° C 20 h |
5 x 105 |
Coagulase positive |
DBPA |
6,4 x 104 |
S. aureus |
TSB |
3.8 x 104 |
MSA |
2.3 x 104 |
|
Coagulase negative |
CBAB 37° C aerobic and anaerobic 18 to 24 h |
nd |
S. epidermidis |
|
nd |
Streptococcus group ẞ |
LIM |
nd |
Streptococcus spp. |
CBAB + SSS |
nd |
Enterococcus spp. |
CBAB + DHB 37° C 18 to 24 h |
4,8x106 |
Pseudomonas spp. |
PIA 35° C 18 to 48 h
|
3,6 x 105 |
* The enumerations of wound-contaminating-bacteria are expressed as means of 5 sampling each of three repetitions
Three replicates were considered. The results were recorded as means ± SD of the duplicate experiment. Differences between means of data were compared by Least Significant Difference (LSD) calculated using the Statistical Analysis System (S.A.S., Institute, Inc. Cary, NC, USA).
The dye (PMA™ Biotium Inc. Hayward, CA, US) is a photoreactive dye with high affinity for DNA. It intercalates into DNA and forms a covalent linkage upon exposure to intense visible light. It is cell membrane impermeable. When a sample comprising both live and dead bacteria is treated with PMA™, only dead cells are susceptible to DNA modification due to their compromised cell membranes. Thus, it permits selective detection of the sole live cells.
DNA extraction was performed using ChargeSwitch® gDNA Mini Bacteria Kit (Life Technologies Italia, Monza, MB, Italy) following manufacturer's instructions.
The molecular identification and characterization was made using specific primer pairs for each isolated bacterium as mentioned in the section microbiological analysis, mixture and reaction conditions as reported in literature (Table 2 list A and list B).
Two primer pairs that amplify specific E. coli 16S rRNA sequences and fourteen primer pairs that specifically amplify target gene coding for virulence factors (adhesins and toxins) were employed to characterize the E. coli isolates [18].
The detection and identification of Staphylococcus spp. were performed by multiplex PCR with primer pairs that amplify variable sequences of the nuc gene, specific for Staphylococcus genus and expressing thermo nuclease enzyme, mixtures and reaction conditions as reported in the literature [19].
Enterococci isolates were identified using Internal Transcribed Spacer region to identify the majority of enterococci and the ddl genes encoding D-alanine-D-alanine ligase specific for the individual species [20,21].
P. aeruginosa is an opportunistic pathogen capable of infecting virtually all tissues. Its molecular detection and identification was performed using PCR amplification of two specific outer bacterial membrane proteins: I lipoprotein (oprI) and L lipoprotein (oprL) using two sets of primers (fPS1/rPS2 and fPAL1/rPAL2) were used in multiplex PCR [22,23].
The amplified products were revealed by electrophoresis in agarose (2%) gel, stained with GelGreen™ Nucleic Acid Gel Stains (Biotium, Inc., Hayward, CA, USA) and visualized by blue light transilluminator UltraBright-LED (Syngene Europe, Cambridge, UK) using a 100 bp DNA ladder (Thermo Fisher Scientific. Milano, Italy).
Water without templates and the reference strains were considered as negative and positive controls.
Target gene |
Primer sequences |
Amplicon (bp) |
1) LT |
f 5'-ATT TAC GGC GTT ACT ATC CTC-3' r 5'-TTT TGG TCT CGG TCA GAT ATG-3' |
281 |
2) STa |
f 5'-TCC GTG AAA CAA CAT GAC GG-3' r 5'-ATA ACA TCC AGC ACA GGC AG-3' |
244 |
3) STb |
f 5'-GCC TAT GCA TCT ACA CAA TC-3' r 5'-TGA GAA ATG GAC AAT GTC CG-3' |
279 |
4) Stx1all |
f 5'-CGC TGA ATG TCA TTC GCT CTG C-3' r 5'-CGT GGT ATA GCT ACT GTC ACC-3' |
302 |
5) Stx2all |
f 5'-CTT CGG TAT CCT ATT CCC GG-3' r 5'-CTG CTG TGA CAG TGA CAA AAC GC-3' |
516 |
6) Stx2e |
f 5-ATG AAG AAG ATG TTT ATA GCG-3' r 5'-TCA GTT AAA CTT CAC CTG GGC-3' |
264 |
7) EAST1
|
f 5'-CCA TCA ACA CAG TAT ATC CGA-3' r 5'-GGT CGC GAG TGA CGG CTT TGT-3' |
111 |
8) eae
|
f 5'-GGA ACG GCA GAG GTT AAT CTGCAG-3' r 5'-GGC GCT CAT CAT AGT CTTTC-3' |
775 |
9) hlyA |
f 5'-AGCTGCAAGTGCGGGTCTG-3' r 5'-TACGGGTTATGCCTGCAAGTTCAC-3' |
569 |
10) F4 (K88) |
f 5'-GCT GCA TCT GCT GCA TCT GGTATG G-3' r 5'-CCA CTG AGT GCT GGTAGT TAC AGC C-3' |
792 |
11) F5 (K99) |
f 5'-TGC GAC TAC CAA TGC TTC TG-3' r 5'-TAT CCA CCA TTA GAC GGA GC-3' |
450 |
12) F6 (P987) |
f 5'-TCT GCT CTT AAA GCT ACT GG-3' r 5'-AAC TCC ACC GTT TGT ATC AG-3' |
333 |
13) F17 |
f 5'-GGG CTG ACA GAG GAG GTG GGGC-3' r 5'-CCC GGC GAC AAC TTC ATCACC GG-3' |
411 |
14) F18 |
f 5'-GTG AAA AGA CTA GTG TTT ATT TC-3' r 5'-CTT GTA AGT AAC CGC GTA AGC-3' |
510 |
15) F41 |
f 5'-GAG GGA CTT TCA TCT TTT AG-3' r 5'-AGT CCA TTC CAT TTA TAG GC-3' |
431 |
16) E16SI |
f 5'-CCCCCTGGACGAAGACTCAC-3' r 5'-ACCGCTGGCAACAAAGGATA -3' |
401 |
17) E16SII |
f 5'-AGAGTTTGATGGCTCAG-3' r 5'-GGACTACCAGGGTATCTAAT-3' |
798 |
Species |
Target gene |
Primer sequences |
Amplicon (bp) |
S. aureus |
nunc 1 |
f 5' - ATG AAG TCA AAT AAA TCG CT- 3' r 5' - TTT GGT GAA AAA TAC TTC TC - 3' |
458 |
S. hyicus |
nunc 2 |
f 5' - AAA AAT AAC AAC AGG ATT GA - 3' r 5' - GTA AAG TCT GAA GCT TCT TT - 3' |
270 |
S. intermedius |
nunc 3 |
f 5 '- GAA AAA AAT TAC AAC AGG CG - 3' r 5' - CAC ATC CGT TGA AGA CTT TT - 3' |
106 |
P. aeruginosa
|
PS
|
f 5' - ATG AAC AAC GTT CTG AAA TTC TCT GCT - 3' r 5' - CTT GCG GCT GGC TTT TTC CAG - 3' |
249 |
PAL
|
f 5' - ATG GAA ATG CTG AAA TTC GGC - 3' r 5' - CTT CTT CAG CTC GA - 3' |
504 |
|
Enterococcus spp. |
ITS |
f 5'-CAA GGC ATC CAC CGT-3' r 5'-GAA GTC GTA ACA AGG-3 |
|
E. faecium |
ddlEfm |
f 5′-AGA GAC ATT GAA TAT GCC -3′ r 5′-CTA ACA TCG TGT AAG CT -3′ |
550 |
E. faecalis |
ddlEfs |
f 5′-ATC AAG TAC AGT TAG TCT -3 r 5′-ACG ATTCAA AGC TAA CTG -3′ |
491 |
10 -15 Different types of E. coli fimbriae
The isolation of goat Peripheral Blood Mononucleate Cells procedure, Peripheral Blood Mononucleate Cells culture conditions, and their quantification by viable cells WST-1 test used in the experiment were those reported by De Matties et al. [27]. Neem oil and Tween 20® were used singularly as controls.
The most interesting feature of the plate concerns the presence of compounds with high fluorescent reaction at between Rf 0.55- 0.66, that are perfectly visible at 366 nm after derivatization with anisaldehyde. These spots can be attributed to compounds with high conjugated unsaturation in polycyclic aromatic structures, very different from those of the nortriterpenes limonoids, so far considered as responsible for the activities investigated in our studies (Figure 1).
faecalis, Staphylococcus aureus and P. aeruginosa were detected. Amplicons of the expected size were obtained from DNA extracted from each bacterium investigated as showed in list A and List B of Table 1.
Amplicons were also obtained from DNA extracted from American Type Culture Collection reference strain of each tested bacterium (positive controls) while amplified products were never obtained from reaction mixtures without templates (negative controls) (Figure 2).
As shown in Table 4, the percent bacterial Growth Reduction revealed at 100 μL, 10 μL, 1 μL and 0.1 μL of neem oil ranged, respectively, 74.51 ± 1.15 - 95.51 ± 1.15; 59.61 ± 0.58 - 75.61 ± 1.00; 48.10 ± 0.10 -61.67± 1.35 and 32.63 ± 1.73 - 32.63 ± 1.73. There was significant difference among the antibacterial activity at the mentioned neem oil dilutions.
The highest percent bacterial Growth Reductions were detected at 100 μL neem oil and they concerned mainly pathogenic E. coli isolates as follows: E. coli hlyA 95.51 ± 1.15, E. coli eae 91.25 ± 1.43, E. coli EAST1 89.65 ± 1.53 (Table 4).
Peripheral Blood Mononucleate Cells viability. This was revealed in Peripheral Blood Mononucleate Cells culture of 24h and 48h incubation periods. Figure 2 C represents the effect of neem oil and Tween®20 (1:1 v/v) on percent viability of Peripheral Blood Mononucleate Cells grew in Roswell Park Memorial Institute medium at different dilutions (1:2x102, 1:2x103, 1:2x104; 1:2 x105; 1:2 x106). If Tween®20 is used with neem oil the adverse effect increases at 1:2 x102 dilution (28% cell viability) while it remains similar at the dilution 1:2 x103 (6% cell viability) respect the treatment with only Tween®20. This was revealed either in Roswell Park Memorial Institute medium culture of 24h or 48 h incubation periods.
There is significant difference between the adverse effect of neem oil, Tween®20, Tween®20 and neem oil treatments at dilution 1:2 x102 on percent Peripheral Blood Mononucleate Cells viability. Furthermore, there is no significant difference between Tween®20 and Tween®20 and neem oil treatments at dilution 1:2 x103. While there is significant difference between neem oil and Tween®20 as well Tween®20 and neem oil treatments at the same dilution (Figure 3).
The phytochemical studies and bio-assay guided fractionation showed that the insecticidal activity are correlated with the tetranortriterpenoids, such as those already mentioned as the
Bacteria |
GIZ (mm) |
|||
Neem oil 100 µL |
Tween®20 100 µL |
Water 100 µL |
Ciprofloxacin 100 µL |
|
E. coli eae |
28.71 ± 1.29 a |
- |
- |
26.52 ± 1.07 b |
E. coli hly A |
29.59 ± 1.17 a |
- |
- |
23.62 ± 1.00 a |
E. coli EAST 1 |
30.21 ± 1.38 a |
- |
- |
24.53 ± 0.67 a |
S. aureus |
23.33 ± 0.53 a |
- |
- |
26.42 ± 0.58 b |
E. faecalis |
26,18 ± 0.81 a |
- |
- |
30.61 ± 1.21 b |
P. aeruginosa |
25.83 ± 1.58 a |
- |
- |
29.61 ± 1.11 b |
E. coli ATCC® 51813™ |
26.33 ± 0.58 a |
- |
- |
26.75 ± 0.82 a |
S. aureus ATCC® 29213™ |
24.07 ± 1.09 a |
- |
- |
27.41 ± 0.76 b |
E. faecalis ATCC® 29212™ |
23.41 ± 1.,06 c |
- |
- |
28.42 ± 0.20 b |
P. aeruginosa ATCC® 27853™ |
22.68 ± 0.55 a |
- |
- |
31.42 ± 1.39 b |
The plates were done in triplicate for each bacterial isolate and the experiment was performed twice. The results were recorded as mean ± S.D. of the duplicate experiment. Mean values with different lowercase letters in the row are significantly different (p ≤ 0.05). Differences between means of data were compared by LSD calculated using the SAS.
Bacteria |
Growth Reduction (%) |
|||
Neem Oil (100 µL) |
Neem Oil (10 µL) |
Neem Oil (1 µL) |
Neem Oil (0.1 µL) |
|
E. coli eae |
91.25 ± 1.43 d |
75.61 ± 1.00 c |
61.67 ± 1.33 b |
41.31 ± 2.08 a |
E. coli hly A |
95.51 ± 1.15 d |
74.70 ± 1.00 c |
51.88 ± 1.33 b |
41.86 ± 1.00 a |
E. coli EAST 1 |
89.65 ± 1.53 d |
68.61 ± 1.00 c |
58.77 ± 1.33 b |
48.51 ± 2.08 a |
S. aureus |
79.65 ± 1.53 d |
59.61 ± 1.00 c |
48.77 ± 1.33 b |
48.51 ± 2.08 a |
E. fecalis |
74.51 ± 1.15 d |
69.70 ± 1.00 c |
44.38 ± 1.33 b |
44.86 ± 1.00 a |
P. aeruginosa |
85.70 ± 1.53 d |
68.73 ± 2.08 c |
48.59 ± 2.00 b |
32.63 ± 1.73 a |
E. coli ATCC® 51813™ |
89.71 ± 1.00 d |
59.61 ± 0.58 c |
49.17 ± 0.00 b |
36.48 ± 0.89 a |
S. aureus subsp. aureus ATCC® 6538™ |
81.51 ± 1.15 d |
59.70 ± 1.00 c |
45.78 ± 1.33 b |
34.86 ± 1.00 a |
E. faecalis ATCC® 7088™ |
89.90 ± 1.00 c |
58.79 ± 1.00 c |
48.10 ± 0.10 b |
35.58 ± 1.20 a |
P. aeruginosa ATCC® 27853™ |
88.90 ± 1.00 d |
58.79 ± 1.00 c |
49.40 ± 0.00 b |
36.68 ± 1.20 a |
Authors carried out dose determination studies and field study on parasiticidal activity of neem oil for the evaluation of its efficacy according the guidelines of World Association for the Advancement of Veterinary Parasitology. The neem oil parasiticidal activity was evaluated towards goat (Capra hircus L.) lice (Linognathus stenopsis and Damalinia caprae). The results confirmed that neem oil is an effective parasiticide that control two lice (Linognathus stenopsis and Damalinia caprae) biological cycles with one neem oil dose administration. Furthermore, they evaluated skin reactions during the experimentation using Draize Scoring System [30] and demonstrated neem oil does not cause irritation of skin. It was always obtained a score equal zero regardless of the dose used (manuscript submitted and under revision). Due the possibility of neem oil absorption through skin, a methodology was set up to evaluate the effect of neem oil on goat blood cells [27]. Neem oil was efficacy in containing louse with no side effects on treated goats.
Many studies carried out so far demonstrated the microbial inhibitory effects of different phytoconstituents of A. indica. Neem has been reported to exhibit antibacterial as well as antifungal activities depending from which part of the plant and how extracts were obtained. Margolone (12-methyl-7-oxopodocarpa-8,11,13-triene-13-carboxylic acid), margolonone (2-Phenanthrenecarboxylicacid, 4b, 5, 6, 7, 8, 8a, 9, 10-octahydro-3, 4b, 8, 8-tetramethyl-7, 10-dioxo-, (4bS,8aR)- ) and isomargolonone ("3-Phenanthrenecarboxylic acid, 4b, 5,6,7,8,8a, 9,10-octahydro- 2,4b, 8,8- tetramethyl-7,10-dioxo-,(4bS-trans)-") are tricyclic diterpenoids isolated from stem bark, nimbidin (Methyl (2R, 3aR, 4aS, 5R, 5aR, 6R, 9aR, 10S, 10aR)-5-(acetyloxy)-2-(furan- 3-yl)-10-(2-methoxy-2-oxoethyl)-1,6,9a,10a-tetramethyl-9- oxo-3,3a,4a,5,5a,6,9,9a,10,10a-decahydro-2H-cyclopenta[b] naphtho[2,3-d]furan-6-carboxylate ) and nimbolide (4α,5, α 6 α,7 α,15,17 α )-7,15:21,23-diepoxy-6-hydroxy-4,8-dimethyl- 1-oxo-18,24-dinor-11,12-secochola-2,13,20,22-tetraene-4,11- dicarboxylic acid α-lactone methyl ester] from seed oil, as well azadirachtins (dimethyl (2aR,3S,4S,R,S,7aS,8S,10R,10aS,10 bR)- 10-(acetyloxy)- 3,5-dihydroxy- 4-[(1S,2S,6S,8S,9R,11S)- 2-hydroxy- 11-methyl- 5,7,10-trioxatetracyclo[6.3.1.02,6.09,11]dodec- 3-en- 9-yl]- 4-methyl- 8-{[(2E)- 2-methylbut- 2-enoyl]oxy} octahydro- 1H-furo[3',4':4,4a]naphtho[1,8-bc]furan- 5,10a(8H)- dicarboxylate), quercetin (3,3',4',5,7-pentahydroxyflavone) and β- sitosterol (17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl- 2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a] phenanthren-3-ol) from leaves, all show several biological activity, among them antimicrobial activity, too [31-35].
This study confirms neem oil antibacterial activity towards wound-contaminating-bacteria resulting from ectoparasites sting and bite, too. Therefore, the neem oil should be considered as a safe and ready to use innovative topical solution for the care of wounds.
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