Antimicrobial Activity of Five Essential Oils against Bacteria and Fungi Responsible for Urinary Tract Infections

Abstract

Urinary tract infections are frequently encountered in small animal practice. Escherichia coli and Enterococcus spp. are the most common agents associated to these infections, even though other bacteria and yeasts, such as Candida albicans and Candida famata, may be involved. In view of the increasing problem of the multi-drug resistance, the aim of this study was to investigate the antimicrobial activity of essential oils obtained from star anise (Illicium verum Hook.f.), basil (Ocimum basilicum L.), origanum (Origanum vulgare L.), clary sage (Salvia sclarea L.) and thymus (Thymus vulgaris L.) against multidrug-resistant strains of Escherichia coli, Enterococcus spp., Candida albicans and Candida famata previously isolated from dogs and cats with urinary tract infections. Enterococci were resistant to Illicium verum and Salvia sclarea, such as Candida to Salvia sclarea. Thymus vulgaris and Origanum vulgare essential oils showed the best activity against all the tested pathogens, so they could be proposed for the formulation of external and/or intravesical washes in small animals.

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Article
Antimicrobial Activity of Five Essential Oils against
Bacteria and Fungi Responsible for Urinary
Tract Infections
Valentina Virginia Ebani 1,2 ,*, Simona Nardoni 1,2, Fabrizio Bertelloni 1ID , Luisa Pistelli 2,3 and
Francesca Mancianti 1,2
1Department of Veterinary Science, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy;
simona.nardoni@unipi.it (S.N.); fabrizio.bertelloni@vet.unipi.it (F.B.); francesca.mancianti@unipi.it (F.M.)
2Centro Interdipartimentale di Ricerca “Nutraceutica e Alimentazione per la Salute”, University of Pisa,
via del Borghetto 80, 56124 Pisa, Italy; luisa.pistelli@unipi.it
3Department of Pharmacy, University of Pisa, via Bonanno 6, 56126 Pisa, Italy
*Correspondence: valentina.virginia.ebani@unipi.it; Tel.: +39-05-0221-6968
Academic Editor: Daniela Rigano
Received: 12 June 2018; Accepted: 5 July 2018; Published: 9 July 2018


Abstract:
Urinary tract infections are frequently encountered in small animal practice. Escherichia coli
and Enterococcus spp. are the most common agents associated to these infections, even though
other bacteria and yeasts, such as Candida albicans and Candida famata, may be involved. In view
of the increasing problem of the multi-drug resistance, the aim of this study was to investigate
the antimicrobial activity of essential oils obtained from star anise (Illicium verum Hook.f.),
basil (Ocimum basilicum L.), origanum (Origanum vulgare L.), clary sage (Salvia sclarea L.) and
thymus (Thymus vulgaris L.) against multidrug-resistant strains of Escherichia coli,Enterococcus spp.,
Candida albicans and Candida famata previously isolated from dogs and cats with urinary tract infections.
Enterococci were resistant to Illicium verum and Salvia sclarea, such as Candida to Salvia sclarea.
Thymus vulgaris and Origanum vulgare essential oils showed the best activity against all the tested
pathogens, so they could be proposed for the formulation of external and/or intravesical washes in
small animals.
Keywords:
Escherichia coli;Enterococcus;Candida; antibiotic resistance; antimycotic resistance;
essential oil
1. Introduction
Infections of the urinary tract are frequent and sometimes can induce severe threat both in human
and veterinary medicine, mostly affecting dogs and cats. Urinary tract infections (UTI) may be localized
to the upper tract (kidney and adjacent ureter) or the lower tract (bladder and adjacent urethra) and
more than one organ is often involved [
1
]. These infections are usually caused by bacteria, mainly
those of the intestinal microflora. Escherichia coli and Enterococcus spp. are the most frequent agents
encountered in UTI cases [
2
–
4
]. Even though infections by haematogenous route are possible, bacteria
usually colonize the genito-urinary tract by ascendant route. In view of the anatomic structure, females
are more prone to UTI than males [1].
Yeasts belonging to Candida genus, in particular Candida albicans, are reported as responsible for
fungal infections of the lower urinary tract, both in dogs and cats [
5
,
6
]. The organism is a commensal of
digestive and genito-urinary tract of both healthy people and animals [
7
]. It can also act as opportunistic
pathogen of skin and mucosae both in receptive animals and in immunocompromised patients [
8
].
Conversely, other Candida species are ubiquitous yeasts and can provoke UTI, when carried by the
Molecules 2018,23, 1668; doi:10.3390/molecules23071668 www.mdpi.com/journal/molecules

Molecules 2018,23, 1668 2 of 12
intestinal content or by environmental contamination. The most severe threat of UTI is the multi-drug
resistance of etiological agents that often negatively affects healing.
Enterococci are of severe concern for their intrinsic antibiotic resistance, particularly to
cephalosporins and aminoglycosides, or acquired resistance to many other antimicrobials [
9
]. E. coli
antibiotic resistance is an increasing problem concerning several antibiotic classes, including the new
antimicrobial agents introduced into clinical medicine [
10
]. Both Enterococci and E. coli present in the
gastrointestinal habitat may acquire antimicrobial resistance genes from other commensal organisms,
transferring them to more pathogenic bacteria [
11
]. The epidemiology of Candida infections has strongly
changed in human patients [
12
], showing an increase of incidence of non-albicans species. Candida spp.
are characterized by marked differences in their antifungal susceptibility pattern [
13
]. Particularly,
C. albicans strains are frequently resistant to azoles [
14
,
15
]). In veterinary medicine information is
scanty, furthermore the choice of molecules allowed for UTI treatment is very limited. Moreover, the
majority of antimycotic drugs administered to human patients represent an off-label drug use.
The spread of drug-resistant pathogens requires novel therapies, so in a view of a natural approach,
the use of essential oils (EOs) to treat urogenital infections has been proposed in human medicine [
16
],
whereas no scientific data about their possible employment in animals are available. EOs are secondary
plant metabolites, obtained by steam or dry distillation or by means of a mechanical treatment from
one single species [
17
]. They are volatile substances, with odorous impact, characterized by different
degrees of antimicrobial activity, in relation to several factors. This property is strictly related to the
pathogen; moreover, it depends on the original plant species, climate conditions, cultivation methods
or harvesting areas, EOs preparation method and EOs composition [17].
Aim of the present study was to investigate the antimicrobial activity of EOs obtained from
star anise (Illicium verum Hook.f.), basil (Ocimum basilicum L.), origanum (Origanum vulgare L.), clary
sage (Salvia sclarea L.) and thymus (Thymus vulgaris L.) against multidrug-resistant strains of E. coli,
Enterococcus spp., C. albicans and against isolates of C. famata characterized by high MIC values for
conventional antimycotic drugs, previously isolated from dogs and cats with UTIs.
2. Results
2.1. Essential oils Analysis
The chemical composition of the tested EOs is reported in Table 1. All the five oils were rich in
monoterpenes. In detail, the main terpenes identified in O. vulgare and T. vulgaris EOs were carvacrol
(65.9%) and thymol (52.6%), respectively, followed by p-cymene (15.3%) only in T. vulgaris. The main
compounds of S. sclarea and O. basilicum were linalyl acetate (54.7%) and linalool (46.0%), respectively.
I. verum EO was mainly composed by the phenylpropanoid anethol (89.8%).

Molecules 2018,23, 1668 3 of 12
Table 1. Relative percentage of the main constituents of tested essential oils.
Class Component RI O.v O.b S.s T.v I.v
2 mh tricyclene 926 1.4
9 mh myrcene 991 2.2
13 mh α-terpinene 1018 2.1
15 mh p-cymene 1026 9.3 15.3
16 mh limonene 1031 3.9
18 om 1,8-cineole 1033 5.9
22 mh γ-terpinene 1062 5.3 2.9
25 om cis-linalool oxide (furanoid) 1074 2.2
28 om trans-linalool oxide (furanoid) 1088 1.8
30 om trans-sabinene idrato 1097 1.8 3.8
31 om linalool 1098 46 8.1
39 om borneol 1165 1.6
41 om 4-terpineol 1177 2.4
43 unknown 1.7
44 pp menthyl chavicol(=estragole) 1195 1.1
46 om thymol methyl ether 1232 1.7
49 om linalyl acetate 1257 54.7
53 pp (E) anethol 1283 89.8
54 om isobornyl acetate 1285 1.6
56 om thymol 1290 52.6
58 om carvacrol 1298 65.9
59 unknown 5.6
60 unknown 7.2
61 om α-limonene diepoxide 1347 8.6
62 pp eugenol 1356 11.5
65 sh β-elemene 1392 2.2
66 sh β-caryophyllene 1418 3.7 6.8
67 sh trans-α-bergamotene 1437 3.6
69 sh α-guaiene 1440 1.1
72 sh germacrene D 1481 3.5
74 sh α-bulnesene 1505 2
75 sh trans-γ-cadinene 1513 2.8
77 sh δ-cadinene 1524 1
80 os caryophyllene oxide 1581 4.8
82 os 1,10-di-epi-cubenol 1614 1
83 os T-cadinol 1640 5.8
87 od scareol 2223 1.3
Legend: RI: retention index measured on HP-5 column; O.v.:Origanum vulgare;O.b.:Ocimum basilicum;S.s.:Salvia
sclarea;T.v.:Thymus vulgaris;I.v.:Illicium verum; mh: monoterpene hydrocarbons; om: oxygenated monoterpenes;
sh: sesquiterpene hydrocarbons; os: oxygenated sesquiterpenes; pp: phenylpropanoids; od: oxygenated diterpenes.
2.2. Antibacterial Activity
Agar Disc Diffusion Method
The results of the agar disc diffusion method testing E. coli and Enterococcus isolates against 21
antibiotics are summarized in Table 2.
All the strains resulted multi-resistant, even though with different resistance patterns.
High percentages of no-sensitive (resistant or intermediate) strains against several antibiotics
were detected, both among the tested E. coli and Enterococcus spp. isolates (Table 3).

Molecules 2018,23, 1668 4 of 12
Table 2.
Results of the agar disc diffusion method testing each Escherichia coli and Enterococcus spp.
isolates with different antibiotics.
Antibiotic
Escherichia coli
Strain n◦
Enterococcus spp.
Strain n◦*
1069 1002 994 986 977 876 835 1091 1079 1034 835 793 654 618 568
ATM R R R I R R S R R R R R R R R
AK S I S R S R S R R R R R I S R
AMC R I R I I R R S S S S S S S S
AMP R R R R R R R R R R I R I I I
KF R R R R R R R R R R R I I S S
CTX I R R S R I S R R R I R R R I
CAZ R R R R R R S R R R R R R R R
CL R R R R R I R R R R R R R I R
CIP R I R R R R R R R R R R S R R
CT R S S R S R S R R R R R R R R
DO R S S R R R R R S I R R S S R
E R R R R R R R I I R R R I S R
ENR R S R R R R R R R R R R I R R
CN R S S S S R R R R I R I S S R
N R I I R R R I R R R R R I I R
PPL R R R R R R R I I R I R I R I
RD I R R I I R I R R I S R S I S
S R I I I I R R R R R R R R I R
STX R S S R R R R S R S S S S S R
TE R S S R R R R R S R R R R R R
TOB R I S R R R R R R R R R S S R
Legend—S: sensitive; I: intermediate; R: resistant; ATM: aztreonam; AK: amikacin; AMC: amoxicillin-clavulanic acid;
AMP: ampicillin; KF: cephalotin; CTX: cefotaxime; CAZ: ceftazidime; CL: cephalexin; CIP: ciprofloxacin; CT: colistin
sulfate; DO: doxycycline; E: erythromycin; ENR: enrofloxacin; CN: gentamicin; N: neomycin; PPL: piperacillin;
RD: rifampicin; S: streptomycin; SXT: sulphametoxazole-trimethoprim; TE: tetracycline; TOB: tobramycin;
*: Enterococcus faecium (strains 1091 ,1079, 1034), Enterococcus faecalis (strains 835, 793, 654, 618), Enterococcus durans
(strain 568).
Table 3.
Number and percentages of Escherichia coli and Enterococcus spp. isolates resulted sensitive,
intermediate or resistant versus tested antibiotics.
Antibiotic Escherichia coli Enterococcus spp.
S (%) I (%) R (%) S (%) I (%) R (%)
ATM 1 (14.3) 1 (14.3) 5 (71.4) 0 0 8 (100)
AK 4 (57.1) 1 (14.3) 2 (28.6) 1 (12.5) 1 (12.5) 6 (75)
AMC 0 3 (42.9) 4 (57.1) 8 (100) 0 0
AMP 0 0 7 (100) 0 4 (50) 4 (50)
KF 0 0 7 (100) 2 (25) 2 (25) 4 (50)
CTX 2 (28.6) 2 (28.6) 3 (42.8) 0 2 (25) 6 (75)
CAZ 1 (14.3) 0 6 (85.7) 0 0 8 (100)
CL 0 1 (14.3) 6 (85.7) 0 1 (12.5) 7 (87.5)
CIP 0 1 (14.3) 6 (85.7) 1 (12.5) 0 7 (87.5)
CT 4 (57.1) 0 3 (42.9) 0 0 8 (100)
DO 2 (28.6) 0 5 (71.4) 3 (37.5) 1 (12.5) 4 (50)
E 0 0 7 (100) 1 (12.5) 3 (37.5) 4 (50)
ENR 1 (14.3) 0 6 (85.7) 0 1 (12.5) 7 (87.5)
CN 4 (57.1) 0 3 (42.9) 2 (25) 2 (25) 4 (50)
N 0 4 (57.1) 3 (42.9) 0 2 (25) 6 (75)
PPL 0 0 7 (100) 0 5 (62.5) 3 (37.5)
RD 0 3 (42.9) 4 (57.1) 3 (37.5) 2 (25) 3 (37.5)
S 0 4 (57.1) 3 (42.9) 0 1 (12.5) 7 (87.5)
STX 2 (28.6) 0 5 (71.4) 6 (75) 0 2 (25)
TE 2 (28.6) 0 5 (71.4) 1 (12.5) 0 7 (87.5)
TOB 0 2 (28.6) 5 (71.4) 2 (25) 0 6 (75)
Legend—S: sensitive; I: intermediate; R: resistant; ATM: aztreonam; AK: amikacin; AMC: amoxicillin-clavulanic acid;
AMP: ampicillin; KF: cephalotin; CTX: cefotaxime; CAZ: ceftazidime; CL: cephalexin; CIP: ciprofloxacin; CT: colistin
sulfate; DO: doxycycline; E: erythromycin; ENR: enrofloxacin; CN: gentamicin; N: neomycin; PPL: piperacillin;
RD: rifampicin; S: streptomycin; SXT: sulphametoxazole-trimethoprim; TE: tetracycline; TOB: tobramycin.

Molecules 2018,23, 1668 5 of 12
Table 4reports the minimum inhibitory concentration (MIC) values, expressed both as percentage
and as mg/mL, testing E. coli and Enterococcus strains against the five selected EOs. I. verum and
S. sclarea EOs did not show any antibacterial activity against Enterococcus isolates, whereas they
induced moderate growth inhibition versus some E. coli strains. Better results have been obtained
by the remaining EOs, mainly O. vulgare and T. vulgaris oils. Results obtained by agar disk diffusion
method and broth microdilution test were comparable. No growth inhibition was observed with the
negative control.
Table 4.
Antibacterial activity expressed as minimum inhibitory concentration (MIC) of the essential
oils against Escherichia coli and Enterococcus strains *.
Bacterial Strain Illicium verum Ocimum basilicum Origanum vulgare Salvia sclarea Thymus vulgaris
% mg/mL % mg/mL % mg/mL % mg/mL % mg/mL
E. coli 1069 0.3 0.611 0.3 0.571 0.15 0.293 0.15 0.279 0.3 0.585
E. coli 1002 0.3
0.611 1.25 2.287 0.6 1.183 1.25 2.232 0.3 0.585
E. coli 994 0.07 0.152 0.15 0.285 0.15 0.293 0.15 0.279 0.07 0.146
E. coli 986 1.25 2.445 NE 0.3 0.587 1.25 2.232 0.3 0.585
E. coli 977 1.25 2.445 1.25 2.287 0.3 0.587 1.25 2.232 0.15 0.292
E. coli 876 0.07 0.152 0.6 1.143 0.15 0.293 0.07 0.139 0.07 0.146
E. coli 835 0.3 0.611 0.6 1.143 0.3 0.587 0.3 0.558 0.07 0.146
Enterococcus 1091 NE 10 18.3 0.6 1.183 NE 1.25 2.342
Enterococcus 1079 NE 2.5 4.575 0.6 1.183 NE 1.25 2.342
Enterococcus 1034 NE 5 9.15 0.6 1.183 NE 0.6 1.171
Enterococcus 835 NE 5 9.15 0.6 1.183 NE 1.25 2.342
Enterococcus 793 NE 1.25 2.287 0.6 1.183 NE 1.25 2.342
Enterococcus 654 NE 1.25 2.287 0.6 1.183 NE 1.25 2.342
Enterococcus 618 NE 2.5 4.575 0.6 1.183 NE 1.25 2.342
Enterococcus 568 NE 5 9.15 0.6 1.183 NE 0.6 1.171
Legend—NE: no effective; *: Enterococcus faecium (strains 1091, 1079, 1034), Enterococcus faecalis (strains 835, 793, 654,
618), Enterococcus durans (strain 568).
2.3. Antimycotic Activity
Selected yeasts showed different patterns of resistance to conventional antimycotic drugs. In detail,
all C. albicans isolates were resistant to voriconazole and to itraconazole, 9/12 to fluconazole, while
MICs yielded from C. famata were high in 3 cases out of 4 for both fluconazole and itraconazole, and in
1/4 case for voriconazole. Caspofungin resulted active for all yeasts isolates.
Selected EOs showed different efficacy against tested yeasts. T. vulgaris EO yielded the lowest
overall MICs and was effective versus all C. famata and versus 11/12 C. albicans isolates with MICs
lower than 1 mg/mL. O. vulgare and O. basilicum appeared less active,even if the lowest MIC among
all tested EOs (0.01%) was obtained by O. vulgare versus a strain of C. albicans. Conversely, S. sclarea
was ineffective versus all examined fungi, at highest concentration tested. I. verum showed the widest
range of activities, resulting completely not effective (>19.52 mg/mL) against four C. albicans isolates
and showing a MIC of 0.19 mg/mL versus two C. famata and one C. albicans. Results in detail of MICs
expressed both as percentage and as mg/mL are reported in Table 5. No apparent relationship among
the profiles of resistance to conventional drugs and MIC values of selected EOs was observed.

Molecules 2018,23, 1668 6 of 12
Table 5. MIC values of selected essential oils, expressed as percentage and weights.
Yeast Strain Ocimum basilicum
%mg/mL
Origanum vulgare
%mg/mL
Salvia sclarea
%mg/mL
Thymus vulgaris
%mg/mL
Illicium verum
%mg/mL
Caspofungin *
mg/L
Voriconazole ◦
mg/L
Itraconazole
mg/L
Fluconazole §
mg/L
C famata1 0.1 0.18 0.1 0.18 >10 >17.86 0.075 0.14 0.1 0.19 0.125 0.25 0.25 2
C famata2 2.5 4.58 1.25 2.25 >10 >17.86 0.075 0.14 1.5 2.93 0.047 0.25 1 16
C famata3 1.5 2.7 1.25 2.25 >10 >17.86 0.075 0.14 1 1.95 0.047 1 1 32
C famata4 0.075 0.13 0.075 0.135 >10 >17.86 0.075 0.14 0.1 0.19 0.125 0.125 1 8
C.albicans1 0.075 0.13 0.075 0.135 >10 >17.86 0.05 0.09 1 1.95 0.125 >32 >32 >256
C.albicans2 0.5 0.9 2 3.6 >10 >17.86 0.5 0.93 >10 >19.56 0.125 >32 >32 >256
C.albicans3 0.075 0.13 0.075 0.135 >10 >17.86 0.075 0.14 0.1 0.19 0.125 >32 >32 >256
C.albicans4 1 1.8 1 1.8 >10 >17.86 0.1 0.19 1.25 2.44 0.125 >32 >32 2
C.albicans5 0.5 0.9 0.1 0.18 >10 >17.86 0.1 0.19 >10 >19.56 0.125 >32 >32 >256
C.albicans6 2.5 4.58 1 1.8 >10 >17.86 0.1 0.19 2 3.9 0.125 >32 >32 >256
C.albicans7 1 1.8 0.075 0.135 >10 >17.86 1 1.87 2 3.9 0.125 >32 >32 0.75
C.albicans8 0.75 1.3 0.1 0.18 >10 >17.86 0.075 0.14 2 3.9 0.094 >32 >32 >256
C.albicans9 0.05 0.09 0.01 0.018 >10 >17.86 0.05 0.09 1 1.95 0.19 >32 >32 >256
C.albicans10 1 1.8 0.75 1.35 >10 >17.86 0.5 0.93 0.5 0.97 0.125 >32 >32 2
C.albicans11 2.5 4.58 0.05 0.09 >10 >17.86 0.05 0.09 >10 >19.56 0.19 >32 >32 >256
C.albicans12 1 1.8 0.1 0.18 >10 >17.86 0.1 0.19 >10 >19.56 0.125 1 >32 >256
Legend—* breakpoint of echinocandins recommended by CLSI for C. albicans up to 0.25 (sensitive), 0.5 (intermediate), from 1 (resistant);
◦
recommended breakpoint by CLSI for C. albicans
up to 0.12 (sensitive), 0.25–0.5 (intermediate), from 1 (resistant); § recommended breakpoint by CLSI for C. albicans up to 2 (sensitive), from 8 (resistant).

Molecules 2018,23, 1668 7 of 12
3. Discussion
The present investigation reports the activity of selected EOs, against multidrug-resistant both
bacterial and fungal organisms causing UTI in pet carnivores. At the best of our knowledge it is the
first study that refers about drug-resistant veterinary isolates of E. coli,Enterococcus spp., C. albicans
and C. famata.
The selected E. coli and Enterococcus spp. strains, previously isolated from dogs and cats with
severe cases of UTI, resulted not sensitive (resistant and intermediate) to several antibiotics. In detail,
one E. coli isolate was not sensitive to any tested antibiotic and some strains were sensitive only to one
or two out of the twenty-one tested antibiotics.
The evaluation of the antibacterial activity of the selected EOs showed promising results, especially
with O. vulgare and T. vulgaris. These EOs, in fact, resulted effective against all the tested isolates
showing MIC values ranging from 0.15% (v/v) (0.293 mg/mL) to 0.6% (v/v) (1.183 mg/mL) for
O. vulgare, and from 0.07% (v/v) (0.146 mg/mL) to 1.25% (v/v) (2.342 mg/mL) for T. vulgaris.
These results are corroborated by previous investigations that found relevant antimicrobial properties
of oregano and thyme EOs related to their main components carvacrol and thymol, but also
other minor constituents such as the monoterpene hydrocarbons
γ
-terpinene and p-cymene [
18
].
I. verum and S. sclarea resulted moderately effective against E. coli strains, whereas no activity was
observed when they were assayed versus Enterococcus spp. isolates. This different activity could
be related to the dissimilar structure of Gram-positive and Gram-negative bacteria cell wall, as
also supposed by
Benmalek et al.
[
19
] who found similar results when tested I. verum EO against
E. coli and Staphylococcus aureus. Activity of I. verum against enterococci is scantly investigated;
however,
Hawrelak et al.
[
20
] found a very weak effectiveness of this EO against Enterococcus faecalis.
About S. sclarea, our results are comparable to those obtained by Frydrysiak et al. [
21
] who found
weak antibacterial activity of Salvia officinalis and Salvia lavandulaefolia, whereas they are totally in
disagreement with other studies in which strong activity of S. officinalis EO was observed against
both Gram-negative bacteria, such as E. coli, and Gram-positive bacteria including enterococci [
22
].
This difference could be related to the original plant species and/or to variability among the
bacterial strains.
Basil EO showed a good antimicrobial activity, with MIC values ranging from 0.15% (v/v)
(0.285 mg/mL) to 1.25% (v/v) (2.287 mg/mL), against all the selected E. coli strains, except for one
isolate (n.986) that showed no-sensitivity to 19 antibiotics among the 21 tested. Very weak activity was
observed against Enterococcus spp. isolates. Antibacterial activity of O. basilicum EO was demonstrated
in other studies that assayed it against multi-drug resistant clinical isolates including E. coli [
23
] and
Enterococcus [
24
]. Interestingly, O. basilicum EO chemical composition reported by Sienkiewicz et al. [
23
]
differed considerably from the EO used in the present study being estragole the main represented
compound (86.4%). Antimicrobial activity of O. basilicum EO is considered mainly related to eugenol
that in our study is present in a moderate amount (11.5%); this could explain the lower MIC values
found with respect to the other EOs.
Even though some among the tested oils did not show a very strong antimicrobial activity, the
comparison to the standard drugs could be significant in some particular cases. For instance, the
overall results obtained with E. coli strain n.876 are interesting, because this isolate was not-sensitive
to all the tested antibiotics, but sensible to all the EOs, with low MIC values. These results suggest
that there is no correlation between the sensitivity to conventional antibacterial drugs and to EOs.
Such natural products could be an alternative when treating some clinical cases.
Although mycotic cystitis is occasionally signaled in carnivores, causative agents investigated
in the present study showed a wide range of resistance to commonly used antimycotic drugs.
Fluconazole represents the preferred drug in the treatment of Candida UTI in human patients [
25
],
while echinocandins and newer azoles are not routinely recommended, due to their low urinary
concentrations [
26
]. Furthermore, itraconazole is the sole antimycotic drug allowed for systemic

Molecules 2018,23, 1668 8 of 12
administration in veterinary medicine, and it is registered for treating feline microsporiasis, only.
In this view, the local application of EOs formulations would be welcome.
T. vulgaris appeared as the most effective EO, probably due to its high content of thymol (52.6%).
This monoterpene compound, together with carvacrol, appeared to be able to completely block
ergosterol synthesis at the MIC values [
27
], making porous the membrane and provoking the yeast cell
killing. Carvacrol is mostly contained in O. vulgare EO (about 66%) that, in the present study, showed
a good efficacy, although with slightly higher MICs when compared to T. vulgaris. Moreover, these
compounds are reported as able to restore antifungal susceptibility to fluconazole in resistant Candida
strains [
28
]. Eugenol is contained in moderate amounts (11.5%) in O. basilicum EO and shares with
thymol the ability to damage cell membrane in C. albicans [
29
]. This EO resulted active against part of
selected yeasts (2/4 C. famata and 5/12 C. albicans). Anethol is the main component (about 90%) of
I. verum EO, and it is reported to have a weak antimicrobial action [30].
Antimicrobial activities of I. verum and its more represented component, anethol, have been
extensively studied, mainly against phytopathogenic fungi [
31
–
34
], indicating a strong antifungal
activity. Antifungal properties of I. verum EO and its main features have been extensively revised
by Wang et al. [
35
], indicating a versatile use of this compound in ethnobotany. At the best of our
knowledge, the only assays of this EO against zoopathogenic fungi are referred to dermatophytes [
36
]
and to some agents of mycotic otitis in pets [
37
]. In both cases I. verum EO showed a poor activity.
Moreover, in the present study it did not yield univocal results, showing wide differences in MIC
values. The marked differences among the results obtained by us, would indicate a yeast individual
sensitivity, considered that, for C. albicans, MIC were ranging from more than 10 to 0.1 mg/mL.
For these reasons individual checking for the
in vitro
efficacy of EOs should be carried out prior to
establish an alternative treatment.
An interesting finding would be the apparent absence of relation between the sensitivity to EOs
with regards to the multidrug resistance.
Topical application of EOs based formulations has been reported in murine models of Candida
vaginitis [
38
,
39
], indicating the feasibility of this route of administration. However, considered the
wide variability of drug/EOs sensitivity patterns among the different isolates of Candida, a preliminary
evaluation of the
in vitro
activity of selected compounds should be performed, to identify the most
suitable EO to treat Candida UTI in pets. This assessment would be advisable, considering that other
non-albicans species such as C. famata,C. kefyr,C. inconspicua,C. rugosa,C. dubliniensis, and C. norvegensis,
although rarely isolated, are now considered emerging species, as their isolation rate has increased
between 2- and 10-fold in human patients over the last 15 years [40].
4. Material and Methods
4.1. Essential Oils
EOs obtained from star anise (I. verum Hook.f.), basil (O. basilicum L.), origanum (O. vulgare L.),
clary sage (S. sclarea L.) and thymus (T. vulgaris L.) were used in the present study. These EOs were
selected for their antibacterial and antifungal actions reported in literature [35,41–43].
All EOs were purchased from the producer (FLORA
®
, Pisa, Italy) and maintained at 4
◦
C in dark
glass vials until used. They were microbiologically analyzed for quality control before antibacterial
and antimycotic tests.
4.2. Gas Chromatography—Mass Spectrometry Analysis
The GC analysis was performed as previously described [44].

Molecules 2018,23, 1668 9 of 12
4.3. Antibacterial Activity
4.3.1. Bacterial Strains
Seven E. coli and eight Enterococcus spp. strains were employed in the study. The strains were
previously isolated from female dogs and cats with urinary tract infections. E. coli and Enterococcus
spp. strains were typed using API 20E and API 20STREP System (BioMérieux, Marcy l’Etoile, France),
respectively and stored in glycerol broth at −80 ◦C.
4.3.2. Agar Disk Diffusion Method
The
in vitro
sensitivity of each E. coli and Enterococcus spp. strain to the following antibiotics
(Oxoid Ltd. Basingstoke, Hampshire, UK) was tested: aztreonam (30
µ
g), amikacin (30
µ
g),
amoxycillin-clavulanic acid (30
µ
g), ampicillin (10
µ
g), cephalothin (30
µ
g), cefotaxime (30
µ
g),
ceftazidime (30
µ
g), cephalexin (30
µ
g), ciprofloxacin (5
µ
g), colistin sulfate (10
µ
g), doxycycline
(30
µ
g), erythromycin (10
µ
g), enrofloxacin (5
µ
g), gentamicin (10
µ
g), neomycin (30
µ
g), piperacillin
(100
µ
g), rifampicin (30
µ
g), streptomycin (10
µ
g), sulphametoxazole-trimethoprim (25
µ
g), tetracycline
(30
µ
g), tobramycin (10
µ
g). The
in vitro
sensitivity to the antibiotics was evaluated by Kirby-Bauer
agar disk diffusion method and the results were interpreted as indicated by the National Committee
for Clinical Laboratory Standards (NCCLS) [45].
Antibacterial activity of each EO was tested by Kirby-Bauer agar disk diffusion method following
the procedures reported by Clinical and Laboratory Standards Institute (CLSI) [
46
] with some
modifications. In details, each EO and mixture was diluted 1:10 in dimethyl sulfoxide (DMSO,
Oxoid Ltd.) and one absorbent paper disk was impregnated with 10
µ
L of each dilution, respectively.
A paper disk impregnated with 10
µ
L of DMSO was included as negative control. All tests were
performed in triplicate.
4.3.3. Minimum Inhibitory Concentration
Minimum inhibitory concentration (MIC) was determined with agar disk diffusion method and
broth microdilution assay. For agar disk diffusion method 10
µ
L of 10%, 5%, 2.5%, 1.25%, 0.62%, 0.31%,
0.15%, 0.07%, 0.03% (v/v) of each EO in DMSO were added on paper disks.
Microdilution assay was performed in 96-well microtitre plates following the protocol previously
described [
43
]. Briefly, the test was carried out in a total volume of 200
µ
L including 160
µ
L of brain
hearth infusion broth (BHIB, Oxoid Ltd.), 20
µ
L of each bacterial suspension and 20
µ
L of each oil
with final EOs concentrations ranging from 10% to 0.03%. Plates were incubated at 37
◦
C for 24 h.
The same assay was performed simultaneously for bacterial growth control (tested bacteria and BHIB)
and sterility control (tested oil or mixture and BHIB). All tests were executed in triplicate. The MIC
value was defined as the lowest concentration, expressed as mg/mL, of EO at which microorganisms
show no visible growth.
4.4. Antimycotic Activity
4.4.1. Fungal Strains
Four strains of C. famata and twelve of C. albicans were used for the assays. All the yeasts were
clinical isolates from female dogs. Identification was achieved using physiological tests such as
cultivation onto Corn Meal Agar (Sigma Aldrich, Milano, Italy) and germ-tube. Microscopy and
biochemical profile evaluated by ID 32 (BioMérieux), were performed, also. When a doubtful profile
was obtained, a final identification was carried out by molecular methods.
4.4.2. Minimal Inhibitory Concentration
Selected EOs were tested by a microdilution assay, as recommended by CLSI M27A
3
for yeasts [
47
],
using dilutions of 10%, 5%, 2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.2%, 0.1%, 0.075%, 0.05%, 0.025%,

Molecules 2018,23, 1668 10 of 12
0.01% and 0.005% to achieve a MIC value. Sensitivity to conventional antimycotic drugs (fluconazole,
voriconazole, itraconazole and caspofungin) was evaluated by Etest (BioMérieux) and breakpoint
values, when available, were calculated following the CLSI recommendations for Candida spp. [48].
5. Conclusions
UTIs are frequent reason for antimicrobial treatment in dogs and cats. Choice of antibiotics and/or
antifungal products should follow the
in vitro
evaluation of causative agent sensitivity. However,
sometimes these microorganisms persist in the urogenital tract and/or a new infection can occur.
The use of EOs has been proposed for the treatment of human UTIs, so these natural products could
be evaluated in veterinary medicine, also, mainly when clinical healing cannot be achieved by using
conventional drugs. Moreover, EOs could be used for relapses prevention. An
in vitro
evaluation
of the isolated pathogens sensitivity to different EOs should be performed to select an oil for UTI
treatment. As reported in literature for other microorganisms, in the present study T. vulgaris and
O. vulgare EOs showed the strongest antimicrobial activity against E. coli,Enterococcus spp., C. albicans
and C. famata, so they could be proposed for the formulation of external and/or intravesical washes,
after a careful evaluation of both cytotoxicity and therapeutic index.
Author Contributions:
Conceptualization, V.V.E., S.N., F.M.; Investigation, V.V.E., S.N., F.B., L.P., F.M.;
Writing—Review & Editing, V.V.E., S.N., F.M.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).