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Citation: Nocera, F.P.; Ferrara, G.;
Scandura, E.; Ambrosio, M.; Fiorito,
F.; De Martino, L. A Preliminary
Study on Antimicrobial Susceptibility
of Staphylococcus spp. and
Enterococcus spp. Grown on Mannitol
Salt Agar in European Wild Boar (Sus
scrofa) Hunted in Campania
Region—Italy. Animals 2022,12, 85.
https://doi.org/10.3390/ani12010085
Academic Editor: Laila Darwich
Soliva
Received: 30 November 2021
Accepted: 28 December 2021
Published: 31 December 2021
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animals
Article
A Preliminary Study on Antimicrobial Susceptibility of
Staphylococcus spp. and Enterococcus spp. Grown on Mannitol
Salt Agar in European Wild Boar (Sus scrofa) Hunted in
Campania Region—Italy
Francesca Paola Nocera 1, * , Gianmarco Ferrara 1, Emanuela Scandura 1, Monica Ambrosio 1,
Filomena Fiorito 1and Luisa De Martino 1,2
1Department of Veterinary Medicine and Animal Production, University of Naples Federico II,
Via F. Delpino 1, 80137 Naples, Italy; gianmarco.ferrara@unina.it (G.F.); e.scandura@studenti.unina.it (E.S.);
monica.ambrosio@unina.it (M.A.); filomena.fiorito@unina.it (F.F.); luisa.demartino@unina.it (L.D.M.)
2Task Force on Microbiome Studies, University of Naples Federico II, 80137 Naples, Italy
*Correspondence: francescapaola.nocera@unina.it; Tel.: +39-081-253-6182
Simple Summary:
During the last decade, an increase in the European wild boar (Sus scrofa) pop-
ulation occurred; thus, over the years, wild boars have become an important potential carrier of
pathogenic bacteria for both livestock animals and pets, but also for humans. Since antibiotic resis-
tance has become one of the greatest challenges of global public health, the aim of the present study
was to define the prevalence and the antibiotic resistance profiles of bacteria grown on the selective
medium mannitol salt agar (MSA), isolated from nasal swabs of wild boars hunted in Campania Re-
gion (southern Italy). The most prevalent isolated bacteria were represented by the
Staphylococcus spp.
and Enterococcus spp. strains, which showed worrying antibiotic-resistant profiles. Consequently,
constant surveillance of wild boars is strongly recommended, in order to assess their role as reservoirs
of antibiotic resistant bacteria and as sentinels of a possible environmental contamination.
Abstract:
The importance of wild boar lies in its role as a bioindicator for the control of numerous
zoonotic and non-zoonotic diseases, including antibiotic resistance. Mannitol Salt Agar (MSA) is a
selective medium used for isolation, enumeration, and differentiation of pathogenic staphylococci.
Other genera such as Enterococcus spp. are also salt tolerant and able to grow on MSA. The present
study focused on the identification, by matrix assisted laser desorption/ionization-time of flight
mass spectrometry (MALDI-TOF-MS), of bacteria grown on MSA isolated from the nasal cavities of
50 healthy wild boars
hunted in Campania Region (southern Italy) in the year 2019. In addition, the
antimicrobial resistance phenotype of the isolated strains was determined by disk diffusion method.
Among genus Staphylococcus, coagulase-negative Staphylococcus (CoNS) were the most common
isolated species, with Staphylococcus xylosus as the most prevalent species (33.3%). Furthermore,
Enterococcus spp. strains were isolated, and Enterococcus faecalis was the species showing the highest
frequency of isolation (93.8%). For staphylococci, high levels of resistance to oxacillin (93.3%)
were recorded. Differently, they exhibited low frequencies of resistance to tested non-
β
-lactams
antibiotics. Among enterococci, the highest resistances were observed for penicillin (93.7%), followed
by ampicillin (75%), and ciprofloxacin (68.7%). Interestingly, 43.7% of the isolated strains were
vancomycin-resistant. In conclusion, this study reports the phenotypic antibiotic resistance profiles
of Staphylococcus spp. and Enterococcus spp. strains isolated from nasal cavities of wild boars hunted
in Campania Region, highlighting that these wild animals are carriers of antibiotic resistant bacteria.
Keywords: Staphylococcus spp.; Enterococcus spp.; antimicrobial resistance; wild boars
Animals 2022,12, 85. https://doi.org/10.3390/ani12010085 https://www.mdpi.com/journal/animals
Animals 2022,12, 85 2 of 12
1. Introduction
The recent focus on veterinary public health aspects in a One Health framework
consisting of game management, with particular reference to wild boars, as reservoirs of
important zoonotic diseases, is of great interest for animal health. Wild boar (Sus scrofa) is
susceptible to numerous viral, bacterial, and parasitic diseases that can have considerable
direct and indirect interest for humans, other populations of wild animals, domestic animals,
the species itself and the environment [
1
]. Furthermore, wild boars are often carrier of
antibiotic-resistant bacteria, favoring their circulation in human, livestock, and natural
environments [
2
]. Particularly, the circulation of antibiotic-resistant staphylococci in natural
ecosystems represents a relevant concern, since these microorganisms can act as vectors of
relevant antimicrobial resistance mechanisms and find a natural reservoir in wild boar [
3
].
Members of the genus Staphylococcus are common colonizers of the skin in mammals [
4
]
and due to their ability to coagulate rabbit plasma, staphylococci have been grouped
into coagulase-positive staphylococci (CoPS) or coagulase-negative staphylococci (CoNS).
Among CoPS, Staphylococcus aureus (S. aureus) represents the main causative agent of
infections such as superficial skin and soft tissue infections, osteomyelitis, and septicemia
both in humans and animals [
5
]. Its medical importance is mainly represented by the
emergence and spread of methicillin-resistant S. aureus (MRSA), which, for its ability to
adapt rapidly to the selective pressure of antibiotics, often presents worrying multidrug
resistance profiles. The distribution of multidrug-resistant MRSA among several apparently
healthy animal species represents a potential worrying public health issue [
6
]. Domestic
swine are frequently colonized by S. aureus, and they are recognized as a main reservoir
for MRSA, but studies on MRSA in wild boars are currently scarce, precise MRSA has
been identified only in wild boars from Germany and Spain [
7
,
8
]. Despite, CoNS have less
virulence factors than S. aureus, they have become important nosocomial pathogens, and
many species colonize the skin and mucous membranes of both humans and animals [
9
].
Moreover, in wildlife animals, the isolation of CoNS has been reported in recent years.
Indeed, Mama et al. [
10
] reported for the first time the isolation of CoNS in Spain with a
percentage of 36.6% from the nose of wild boars, with a high prevalence of species such
Staphylococcus sciuri (S. sciuri) (64/161), Staphylococcus xylosus (S. xylosus) (21/161), and
Staphylococcus chromogenes (S. chromogenes) (17/161). Furthermore, the isolated species
presented antibiotic resistance profiles, with 22.4% showing resistance to at least one
antibiotic, and carrying relevant antibiotic resistant genes such as mecA and tetK genes [
10
].
Multidrug- and methicillin-resistant S. sciuri strains were isolated from wild Ungulates in
Spain [
11
], highlighting the role of these animals as reservoirs of multi-resistant methicillin-
resistant Staphylococcus strains. So, according to literature, the spread of methicillin and
multidrug-resistant CoNS has increased over the years, becoming a worrying threat both
for human and veterinary medicine [
12
–
16
]. In this scenario, CoNS of both human and
veterinary origin represent a great concern, since it is well known their role as reservoirs of
antibiotic resistance genes for other pathogenic bacteria such as S. aureus, increasing their
virulence potential and making them therapeutic challenges [9].
Mannitol salt agar (MSA) plates are generally used for Staphylococcus spp. strains
isolation [
17
], but its high concentration of salt (NaCl) is also tolerated by Enterococcus spp.
and Micrococcaceae [
18
–
20
]. Quiloan et al. [
21
] reported that Enterococcus faecalis (E. faecalis)
was a mannitol positive strain producing, thus, yellow colonies on MSA; differently from
Enterococcus faecium (E. faecium), which lacked this phenotype.
Bacteria of the genus Enterococcus, which are considered harmless commensal in
healthy subjects, are often resistant to a number of clinically important antibiotics, and
therefore are hired as sentinel microorganisms for tracking trends in resistance to antimicro-
bials with Gram-positive activity [
22
]. Enterococci, lactic acid bacteria (LAB), comprise both
pathogenic and commensal microorganisms ubiquitous in environment, in fact they can be
detected from soil, water, plants, wild animals, birds, and insects, even as gut symbionts.
Mostly two subspecies are of particular relevance: E. faecalis and E. faecium. Clinical isolates
Animals 2022,12, 85 3 of 12
of E.faecalis and E. faecium have resistance to many commonly used antimicrobial agents,
such as ampicillin and vancomycin [23].
Moreover, enterococci seem to be less virulent in nature than S. aureus or Gram-
negative bacteria, but vancomycin-resistant Enterococcus (VRE) can cause a variety of
infections and represents a pathogen of growing concern; in fact, in recent years, an
increase in invasive VRE human infections has been reported worldwide [
24
]. However,
like MRSA, VRE is endemic in hospital settings [
25
] and it is becoming difficult to manage,
even though VRE is one of the first documented antibiotic resistant bacteria with primary
origin in animal farming [26].
Different bacterial populations are present within the nasal cavities of wild boars.
The aim of this study was to assess if wild boars could represent a potential risk for other
animals, humans, and the environment, as a source of multidrug-resistant strains able to
grow on MSA medium.
2. Materials and Methods
2.1. Ethical Approval
This study did not involve the use of living wild boars, thus ethical approval was not
required. All nasal swabs were collected from wild boars during the hunting season by
licensed and specialized hunters. Furthermore, before the opening of the hunting season
of the year 2019, hunters trained for a proper sampling of wild boar nasal cavities. After
sampling, swabs were held at 4 ◦C during the transport to the Microbiology laboratory.
2.2. Sample Collection
Nasal swabs from apparently healthy 50 wild boars were collected at the time of
capture in the hunting season. Samples were collected from both male and female wild
boars, weighing between 20 kg and 100 kg. The specific wild boar hunting area was
the one located in the province of Salerno in Campania Region (southern Italy). The
hunt was conducted by authorized hunter teams during the normal hunting period (from
1 October 2019 to 31 December 2019).
The sampling was performed by inserting and rotat-
ing a single swab per animal in both nostrils. Each swab was then placed in Stuart W/O
CH
(Aptaca Spa,
Asti, Italy) transport medium and transferred within 48 h, by using an
icebox, to the laboratory for bacteriological examination.
2.3. Bacterial Isolation and Identification
Nasal swab samples were streaked onto mannitol salt agar (MSA) (Liofilchem Srl,
Teramo, Italy) and incubated aerobically at 37
◦
C for 24 h, in order to collect bacterial
isolates able to grow on this medium. Colonies were firstly identified by standard, rapid
screening techniques: colony morphology, presence, or absence of mannitol fermentation
on MSA, Gram-staining, catalase reaction, and for staphylococci also the tube coagulase
reaction (Oxoid, Ltd., Hampshire, UK) was performed.
Single colonies were subcultured on Columbia Sheep Blood agar (Liofilchem Srl,
Teramo, Italy) and after incubation at 37
◦
C for 24 h, the isolates were identified using the
matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-
MS) (Bruker Daltonics Inc., Bremen, Germany).
Only staphylococci and enterococci isolates were preserved in 16% v/vglycerol broth
and in Microbank tubes (Pro-Lab Diagnostics, Round Rock, TX, USA) at
−
80
◦
C for
further investigation.
2.4. Antibiotyping of Staphylococcus spp. and Enterococcus spp.
Antibiotic resistance profiles of the recovered isolates were evaluated by disk diffu-
sion method on Mueller Hinton agar plates (Liofilchem, Teramo, Italy). All the isolated
staphylococci and enterococci were tested for their susceptibility to the following antibi-
otics: amoxicillin-clavulanate (AUG, 20/10
µ
g), ampicillin (AMP, 10
µ
g), ciprofloxacin (CIP,
5µg),
erythromycin (E, 15
µ
g), gentamicin (CN, 10
µ
g), imipenem (IMI, 10
µ
g), penicillin
Animals 2022,12, 85 4 of 12
(P, 10 IU), sulfamethoxazole-trimethoprim (SXT, 1.25/23.75
µ
g), tetracycline (TE,
30 µg),
and vancomycin (VA, 30
µ
g). Staphylococci were tested also for cefotaxime (CTX,
30 µg),
cefoxitin (FOX, 30
µ
g), cephalothin (KF, 30
µ
g), clindamycin (CD, 2
µ
g), enrofloxacin (ENR,
5
µ
g), and oxacillin (OX, 1
µ
g). The tested antibiotic belonged to 10 different classes.
The antimicrobial susceptibility testing results were interpreted according to the Clinical
and Laboratory Standards Institute guidelines [
27
] and to the European Committee on
Antimicrobial Susceptibility Testing [
28
]. Furthermore, isolates were classified as multidrug-
resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) strains
according to Magiorakos et al. [29].
3. Results
3.1. Bacterial Strains Isolated on MSA
During the hunting season of the year 2019, 50 nasal swabs streaked on MSA and
microbiologically analyzed. Precisely, 37 (74%) swabs yielded Gram-positive isolates,
while 13 (26%) nasal swabs did not yield any Gram-positive bacteria growth. From all
positive processed specimens, pure cultures were detected in 32 nasal swabs while in 5
of them, the co-presence of two different bacterial species was detected. Altogether a
total of 42 bacterial strains was isolated. Specifically, 38 (90.5%) of the isolated strains
resulted to be fermenting mannitol, by forming yellow colonies on MSA, whilst 4 (9.5%)
strains were mannitol-negative by producing light pink colonies. Catalase test, which
is essential to differentiate Staphylococcus spp. (catalase positive) from Enterococcus spp.
(catalase negative), gave positive results for 26 bacterial strains, the remaining 16 isolates
turned out to be negative instead. All the recovered strains were negative to oxidase test.
Furthermore, for the 15 Gram-positive cocci seen in tetrads and clusters on the smear, the
performed staphylocoagulase tests resulted to be all negative, pointing out the absence
of coagulase-positive staphylococci like S. aureus. All the above-mentioned results are
reported in Table 1.
Table 1. Phenotypic features of bacteria isolated on MSA from wild boar nasal swabs.
ID D-Mannitol Fermentation Catalase Test Oxidase Test Staphylocoagulase Test
2 + * + −*−
7 + − − NA **
9 + − − NA
10 + − − NA
11 + − − NA
12 + − − NA
13 + − − NA
14 + − − NA
17 + − − NA
18 −+− −
19 + + −NA
20 + + − −
21 + + −NA
22 + + − −
23 + + − −
25 + − − NA
27 + + −NA
28 + + −NA
29 + + −NA
30 + − − NA
31a + − − NA
31b + − − NA
32 −+−NA
33 + + −NA
Animals 2022,12, 85 5 of 12
Table 1. Cont.
ID D-Mannitol Fermentation Catalase Test Oxidase Test Staphylocoagulase Test
34 −+− −
35 + − − NA
36 + + − −
37 + − − NA
38 + − − NA
39a + + −NA
39b + − − NA
40a + + − −
40b + + − −
43 + + −NA
44 + + − −
45a + + − −
45b + + −NA
46 + − − −
47a + + −NA
47b + + − −
48 + + − −
49 −+− −
* +: positive; * −: negative; ** NA: not applicable.
3.2. Identification of Bacterial Species Recovered from Wild Boars
From a total of 42 Gram-positive isolates, 15 belonged to Staphylococcus spp. (35.7%)
and were all CoNS, whereas 16 belonged to Enterococcus spp. (38.1%) (Figure 1). However,
on MSA, we detected the growth of other Gram-positive bacteria such as Macrococcus spp.
(9.5%; 4/42 strains) and Bacillus spp. (16.7%; 7/42 strains), which were identified with
lower frequency values as represented in Figure 1.
Animals2022,12,xFORPEERREVIEW5of12
32− +− NA
33++− NA
34− +− −
35+−−NA
36++− −
37+−−NA
38+−−NA
39a++− NA
39b+−−NA
40a++− −
40b++− −
43++− NA
44++− −
45a++− −
45b++− NA
46+−−−
47a++− NA
47b++− −
48++− −
49− +− −
*+:positive;*‐:negative;**NA:notapplicable.
3.2.IdentificationofBacterialSpeciesRecoveredfromWildBoars
Fromatotalof42Gram‐positiveisolates,15belongedtoStaphylococcusspp.(35.7%)
andwereallCoNS,whereas16belongedtoEnterococcusspp.(38.1%)(Figure1).However,
onMSA,wedetectedthegrowthofotherGram‐positivebacteriasuchasMacrococcusspp.
(9.5%;4/42strains)andBacillusspp.(16.7%;7/42strains),whichwereidentifiedwith
lowerfrequencyvaluesasrepresentedinFigure1.
Figure1.FrequencyofisolationofGram‐positivebacteriagrownonMSA.
AmongisolatedCoNS,fivedifferentspecieswereidentifiedintotal,asreportedin
Table2.S.xylosusresultedtobethemostprevalentspecies(33.3%;5/15strains),followed
byS.chromogenes(26.7%;4/15strains),Staphylococcushyicus(S.hyicus)(20%;3/15strains),
S.sciuri(13.3%;2/15strains),andStaphylococcussimulans(S.simulans)(6.7%;1/15strains)
(Table2);whereasamongenterococci,E.faecalis,knownforitswidetolerancetohighcon‐
centrationsofsaltandabletofermentmannitol,wasthemostpredominantspecies
Figure 1. Frequency of isolation of Gram-positive bacteria grown on MSA.
Among isolated CoNS, five different species were identified in total, as reported in
Table 2.S. xylosus resulted to be the most prevalent species (33.3%; 5/15 strains), followed
by S. chromogenes (26.7%; 4/15 strains), Staphylococcus hyicus (S. hyicus) (20%; 3/15 strains),
S. sciuri (13.3%; 2/15 strains), and Staphylococcus simulans (S. simulans)(6.7%; 1/15 strains)
(Table 2); whereas among enterococci, E. faecalis, known for its wide tolerance to high con-
centrations of salt and able to ferment mannitol, was the most predominant species (93.8%;
15/16 strains). The other enterococcal isolate was represented by
Enterococcus casseliflavus
(E. casseliflavus) (6.2%; 1/16 strains) showing the same phenotype on MSA.
Animals 2022,12, 85 6 of 12
Furthermore, from five wild boar nasal samples two different bacterial species were
recovered. Precisely, the following co-present species were found per animal (sample ID):
Bacillus megaterium/E. faecalis (39a/39b); S. sciuri/S. xylosus (40a/40b);
S. chromogenes/
Bacillus licheniformis (45a/45b); S. hyicus/S. chromogenes (47a/47b), while from the nasal
swab of one animal, two isolates with different colony morphology were identified as
E. faecalis (31a/31b).
All the strains isolated on MSA were identified by MALDI-TOF-MS with good (log)
scores (1.9 ≤x≥2.0) (Table S1 in Supplementary Data).
3.3. Phenotypic Antibiotic Resistant Profiles of Staphylococcus spp. and Enterococcus spp. Isolates
In this study, the antibiotic resistance profiles of CoNS and Enterococcus spp. strains
were evaluated. The antibiotic resistance profiles of Macrococcus spp. and Bacillus spp.
isolates were not analyzed, due to the low number of the collected isolates.
Both CoNS and Enterococcus spp. strains showed relevant resistance frequencies to the
selected antibiotics. As reported in Figure 2, for CoNS interesting levels of resistance were
observed for
β
-lactam antibiotics. Particularly, the highest levels of resistance were recorded
for oxacillin together with penicillin and ampicillin with 14/15 strains resistant to these
antibiotics (93.3%). Differently, high susceptibility levels were detected for cefoxitin (86.7%;
13/15 strains). Surprisingly, CoNS exhibited low frequencies of resistance to tested non-
β
-lactams antibiotics with values never higher than 20% for ciprofloxacin, erythromycin,
enrofloxacin, gentamicin, and tetracycline, whereas a slightly higher level of resistance
was recorded for clindamycin (40%; 6/15 strains). It is worth noting that resistance to
vancomycin was detected in three CoNS strains (20%). No resistance was recorded for the
antibiotic sulfamethoxazole-trimethoprim (Figure 2). One isolate, identified as S. hyicus
(ID 49), resulted susceptible to all tested antibiotics (Table 2).
According to the classification given by Magiorakos et al. [
29
], 40% (6/15) of CoNs
strains (ID 18, 20, 23, 34, 36, 47b) MDR strains were detected. Moreover, the isolate
S. xylosus
(ID 22) was identified as XDR, showing resistance to at least one antimicrobial
agent of each tested antibiotic classes. No PDR CoNS were isolated. CoNS antibiotic
resistance profiles, and the categorization in MDR and XDR strains are summarized in
Table 2.
Animals2022,12,xFORPEERREVIEW6of12
(93.8%;15/16strains).TheotherenterococcalisolatewasrepresentedbyEnterococcuscas‐
seliflavus(E.casseliflavus)(6.2%;1/16strains)showingthesamephenotypeonMSA.
Furthermore,fromfivewildboarnasalsamplestwodifferentbacterialspecieswere
recovered.Precisely,thefollowingco‐presentspecieswerefoundperanimal(sampleID):
Bacillusmegaterium/E.faecalis(39a/39b);S.sciuri/S.xylosus(40a/40b);S.chromogenes/Ba‐
cilluslicheniformis(45a/45b);S.hyicus/S.chromogenes(47a/47b),whilefromthenasalswab
ofoneanimal,twoisolateswithdifferentcolonymorphologywereidentifiedasE.faecalis
(31a/31b).
AllthestrainsisolatedonMSAwereidentifiedbyMALDI‐TOF‐MSwithgood(log)
scores(1.9≤x≥2.0)(TableS1inSupplementaryData).
3.3.PhenotypicAntibioticResistantProfilesofStaphylococcusspp.andEnterococcusspp.
Isolates
Inthisstudy,theantibioticresistanceprofilesofCoNSandEnterococcusspp.strains
wereevaluated.TheantibioticresistanceprofilesofMacrococcusspp.andBacillusspp.iso‐
lateswerenotanalyzed,duetothelownumberofthecollectedisolates.
BothCoNSandEnterococcusspp.strainsshowedrelevantresistancefrequenciesto
theselectedantibiotics.AsreportedinFigure2,forCoNSinterestinglevelsofresistance
wereobservedforβ‐lactamantibiotics.Particularly,thehighestlevelsofresistancewere
recordedforoxacillintogetherwithpenicillinandampicillinwith14/15strainsresistant
totheseantibiotics(93.3%).Differently,highsusceptibilitylevelsweredetectedforcefox‐
itin(86.7%;13/15strains).Surprisingly,CoNSexhibitedlowfrequenciesofresistanceto
testednon‐β‐lactamsantibioticswithvaluesneverhigherthan20%forciprofloxacin,
erythromycin,enrofloxacin,gentamicin,andtetracycline,whereasaslightlyhigherlevel
ofresistancewasrecordedforclindamycin(40%;6/15strains).Itisworthnotingthatre‐
sistancetovancomycinwasdetectedinthreeCoNSstrains(20%).Noresistancewasrec‐
ordedfortheantibioticsulfamethoxazole‐trimethoprim(Figure2).Oneisolate,identified
asS.hyicus(ID49),resultedsusceptibletoalltestedantibiotics(Table2).
AccordingtotheclassificationgivenbyMagiorakosetal.[29],40%(6/15)ofCoNs
strains(ID18,20,23,34,36,47b)MDRstrainsweredetected.Moreover,theisolateS.xy‐
losus(ID22)wasidentifiedasXDR,showingresistancetoatleastoneantimicrobialagent
ofeachtestedantibioticclasses.NoPDRCoNSwereisolated.CoNSantibioticresistance
profiles,andthecategorizationinMDRandXDRstrainsaresummarizedinTable2.
Figure2.AntimicrobialresistanceprofilesofCoNSisolatedfromwildboars.Antibiotics:AUG:
amoxicillin‐clavulanate;AMP:ampicillin;KF:cephalothin;CTX:cefotaxime;FOX:cefoxitin;CIP:
Figure 2.
Antimicrobial resistance profiles of CoNS isolated from wild boars. Antibiotics: AUG:
amoxicillin-clavulanate; AMP: ampicillin; KF: cephalothin; CTX: cefotaxime; FOX: cefoxitin; CIP:
ciprofloxacin; ENR: enrofloxacin; E: erythromycin; CN: gentamicin; IMI: imipenem; OX: oxacillin; P:
penicillin; SXT: sulfamethoxazole-trimethoprim; TE: tetracycline; VA: vancomycin.
Animals 2022,12, 85 7 of 12
Table 2. Antibiotic resistance phenotype of isolated CoNS strains.
ID CoNs Identified Species Antibiotic Resistance Profiles MDR XDR
2S. sciuri AMP, CIP, OX, P
18 S. chromogenes AMP, KF, CD, E, OX, P, VA X **
20 S. chromogenes AMP, CD, CN, OX, P X
22 S. xylosus AUG, AMP, KF, CTX, FOX, CIP, CD,
ENR, E, CN, IMI, OX, P, TE, VA X
23 S. xylosus AMP, KF, OX, P X
34 S. simulans AMP, KF, CD, OX, P, VA X
36 S. xylosus AUG, AMP, CTX, FOX, CD, IMI, OX, P X
40a S. sciuri AMP, OX, P
40b S. xylosus AMP, OX, P
44 S.hyicus AMP, OX, P
45a S. chromogenes AMP, OX, P
47a S. hyicus AMP, OX, P
47b S. chromogenes AMP, CD, CN, OX, P X
49 S. hyicus —– *
* Sample ID 49 is susceptible to all tested antibiotics. ** X: presence of MDR or XDR phenotype. Antibiotics: AUG:
amoxicillin-clavulanate; AMP: ampicillin; KF: cepalothin; CTX: cefotaxime; FOX: cefoxitin; CIP:ciprofloxacin; ENR:
enrofloxacin; E: erythromycin; CN: gentamicin; IMI: imipenem; OX: oxacillin; P: penicillin; SXT: sulfamethoxazole-
trimethoprim; TE: tetracycline; VA: vancomycin.
The antibiotic resistance profiles of the isolated enterococci are exposed in Figure 3. Re-
sistance to penicillin was found in almost all of the analyzed samples (93.7%;
15/16 strains).
It should be also noted that more than half of the isolates were also resistant to ampicillin
and amoxicillin-clavulanic acid, which presented the same frequency of resistance (75%;
12/16 strains), followed by ciprofloxacin (68.7%) (Figure 3). Interesting levels of resistance
were observed also for gentamicin and erythromycin equally (56.2%; 9/16 strains), whilst
lower values of resistance were recorded for tetracycline and imipenem with 6/16 resistant
strains (37.5%). Resistance to vancomycin was detected, in 7 of 16 total isolates (43.7%)
as represented in Figure 3. Intriguingly, from a wild boar nasal swab two isolated of
the same species E. faecalis were recovered, showing a different antimicrobial resistance
phenotype. Precisely, ID 31b-E. faecalis isolate appeared to be a MDR strain, the other
E. faecalis (ID 31a) strain resulted to be more susceptible, with only two phenotypic re-
sistances as reported in Table 3. As for CoNS, also for enterococci no resistance against
sulfamethoxazole-trimethoprim was observed. However, the overall prevalence of antibi-
otic resistance among Enterococcus spp. isolates evaluated in the present study emerged to
be high; 62.5% of isolated enterococci (10/16) were MDR strains, while 25% (4/16) were
identified as XDR isolates. Pandrug resistance was not detected. Antibiotic resistance pro-
files of isolated enterococci, and their categorization in MDR and XDR strains are indicated
in Table 3.
Animals 2022,12, 85 8 of 12
Animals2022,12,xFORPEERREVIEW8of12
Figure3.AntibioticresistanceprofilesofEnterococcusspp.strainsisolatefromwildboars.Antibiot‐
ics:AUG:amoxicillin‐clavulanate;AMP:ampicillin;CIP:ciprofloxacin;E:erythromycin;CN:gen‐
tamicin;IMI:imipenem;P:penicillin;SXT:sulfamethoxazole‐trimethoprim;TE:tetracy‐cline;VA:
vancomycin.
Table3.PhenotypicantibioticresistanceprofilesofcollectedEnterococcusspp.strains.
IDEnterococcusSpeciesAntibioticResistanceProfilesMDRXDR
7E.faecalisAUG,AMP,CIP,IMI,PX*
9E.faecalisAUG,AMP,CIP,E,CN,IMI,PX
10E.faecalisAUG,AMP,CIP,E,CN,IMI,P,TE,VAX
11E.faecalisAUG,AMP,CIP,E,CN,IMI,PX
12E.faecalisAUG,AMP,E,CN,PX
13E.casseliflavusCIP,IMI,PX
14E.faecalisCIP,IMI,PX
17E.faecalisAUG,AMP,CIP,IMI,P,TEX
25E.faecalisAUG,AMP,CIP,E,CN,P,VAX
30E.faecalisAUG,AMP,CIP,E,P,VAX
31aE.faecalisP,TE
31bE.faecalisAUG,AMP,CIP,CN,P,TE,VAX
35E.faecalisAUG,AMP,E,P,VAX
37E.faecalisAUG,AMP,CIP,E,CN,P,TE,VAX
38E.faecalisE,CN
39bE.faecalisAUG,AMP,CN,P,TE,VAX
*X:presenceofMDRorXDRphenotype.Antibiotics:AUG:amoxicillin‐clavulanate;AMP:ampi‐
cillin;CIP:ciprofloxacin;E:erythromycin;CN:gentamicin;IMI:imipenem;P:penicillin;SXT:sul‐
famethoxazole‐trimethoprim;TE:tetracycline;VA:vancomycin.
4.Discussion
Duringthelastdecade,anincreasingoftheEuropeanwildboarpopulationhasoc‐
curred;thus,overtheyearswildboarshavebecomeanimportantpotentialsourceof
pathogenicbacteriaforbothlivestockanimalsandpets,especiallyhuntingdogs,butalso
forhumans,duetoamajorconsumptionofwildboarmeatlinkedtogrowinghunting
activities[1].InItaly,therearemanyreportsonthereservoirroleplayedbywildboars
forzoonoticbacteriasuchasBrucellaspp.,Mycobacteriumspp.,andSalmonellaspp.,which
oftenshowworryingantibioticresistanceprofiles[30–34].Antibioticresistanceiscur‐
rentlyoneofthegreatestchallengesofglobalpublichealth.Globally,acontinuedincrease
inthenumberofresistantbacteriaisconstantlyrecorded,inbothhumansandanimals
Figure 3.
Antibiotic resistance profiles of Enterococcus spp. strains isolate from wild boars. Antibiotics:
AUG: amoxicillin-clavulanate; AMP: ampicillin; CIP: ciprofloxacin; E: erythromycin; CN: gentamicin;
IMI: imipenem; P: penicillin; SXT: sulfamethoxazole-trimethoprim; TE: tetracy-cline; VA: vancomycin.
Table 3. Phenotypic antibiotic resistance profiles of collected Enterococcus spp. strains.
ID Enterococcus Species Antibiotic Resistance Profiles MDR XDR
7E. faecalis AUG, AMP, CIP, IMI, P X *
9E. faecalis AUG, AMP, CIP, E, CN, IMI, P X
10 E. faecalis AUG, AMP, CIP, E, CN, IMI, P, TE, VA X
11 E. faecalis AUG, AMP, CIP, E, CN, IMI, P X
12 E. faecalis AUG, AMP, E, CN, P X
13 E. casseliflavus CIP, IMI, P X
14 E. faecalis CIP, IMI, P X
17 E. faecalis AUG, AMP, CIP, IMI, P, TE X
25 E. faecalis AUG, AMP, CIP, E, CN, P, VA X
30 E. faecalis AUG, AMP, CIP, E, P, VA X
31a E. faecalis P, TE
31b E. faecalis AUG, AMP, CIP, CN, P, TE, VA X
35 E. faecalis AUG, AMP, E, P, VA X
37 E. faecalis AUG, AMP, CIP, E, CN, P, TE, VA X
38 E. faecalis E, CN
39b E. faecalis AUG, AMP, CN, P, TE, VA X
* X: presence of MDR or XDR phenotype. Antibiotics: AUG: amoxicillin-clavulanate; AMP: ampicillin; CIP:
ciprofloxacin; E: erythromycin; CN: gentamicin; IMI: imipenem; P: penicillin; SXT: sulfamethoxazole-trimethoprim;
TE: tetracycline; VA: vancomycin.
4. Discussion
During the last decade, an increasing of the European wild boar population has
occurred; thus, over the years wild boars have become an important potential source of
pathogenic bacteria for both livestock animals and pets, especially hunting dogs, but also
for humans, due to a major consumption of wild boar meat linked to growing hunting
activities [
1
]. In Italy, there are many reports on the reservoir role played by wild boars
for zoonotic bacteria such as Brucella spp., Mycobacterium spp., and Salmonella spp., which
often show worrying antibiotic resistance profiles [
30
–
34
]. Antibiotic resistance is currently
one of the greatest challenges of global public health. Globally, a continued increase in
the number of resistant bacteria is constantly recorded, in both humans and animals [
35
].
This scenario is generally linked to an improper use of antibiotics in veterinary and human
clinical settings, which results in an environmental contamination. Consequently, wildlife
animals, particularly wild ungulates, and wild birds, are becoming reservoirs and spreaders
Animals 2022,12, 85 9 of 12
of multidrug-resistant bacteria [
10
,
11
,
14
], and it is cause of great concern, since these
free-living animals are not subjected to antibiotic pressure. However, information on
antibiotic resistant profiles of opportunistic pathogens such as staphylococci and enterococci
isolated from wild boars are still scarce both in Italy and worldwide. To the best of our
knowledge, this is the first study in Italy on the prevalence and antibiotic resistance
phenotype of Staphylococcus spp. and Enterococcus spp. recovered from nasal samples of
wild boars in southern Italy. All 50 nasal swabs, collected in the hunting season 2019, were
streaked on MSA. Although MSA is a selective and differential medium generally used
for staphylococci isolation, in the present study, not only the growth of CoNS, but also of
E. faecalis and
E. casseliflavus
strains was detected. This latter obtained result agrees with
Quiloan et al. [
21
], who already reported enterococci ability to grow on MSA, since they are
salt tolerant. Furthermore, in this study the isolation of some strains of
Macrococcus spp.,
genus closely related to genus Staphylococcus consisting of eleven species typically found
as commensals in a range of animal hosts [
36
], and Bacillus spp. were observed, but with
a lower prevalence than CoNS and enterococci. It is worth of noting that almost all the
isolated strains resulted to be mannitol positive (90.5%; 38/42 strains); the remaining four
strains were not able to ferment mannitol (9.5%). Specifically, all E. faecalis strains but
also
E. casseliflavus
isolate fermented mannitol on MSA, according to the data described by
Quiloan et al. [
21
]. Referring to CoNS, 12 isolated were mannitol fermenting, whilst a strain
of each of the following species: S. chromogenes,S. hyicus, and S. simulans grew by forming
light pink colonies on MSA. According to literature, CoNS ability to ferment mannitol on
MSA vary among different species and strains of the same species [
37
–
39
]. The percentage
of CoNS was of 35,8% and it is not surprising since CoNS are normal inhabitants of skin
and mucosal membranes of animals and humans. Moreover, our positivity percentage is
similar to the one recorded by Mama et al. [
10
], which reported a prevalence value of 37.7%
for CoNS isolated from nasal swabs of wild boars hunted in Spain in 2016. Differently
from Mama et al. [
10
], herein no coagulase positive staphylococci (CoPS) were isolated,
even though wild boar is considered one of the main S. aureus reservoirs among wild
animals [
40
,
41
]. Despite CoNS have long been considered less pathogenic than S. aureus
and other CoPS, CoNS infections have become increasingly difficult to treat for the increased
number of circulating multidrug-resistant strains in recent years [
42
]. In this study, CoNS
displayed interesting resistance profiles. Particularly, CoNS showed the highest levels of
resistance to oxacillin together with penicillin and ampicillin with 14/15 strains resistant
equally to these antibiotics (93.3%), whilst low levels of resistance were recorded for
cefoxitin (13.3%). Interestingly, low frequencies of resistance (<50%) were observed for
the tested non-
β
-lactams antibiotics, such as ciprofloxacin, clindamycin erythromycin,
enrofloxacin, gentamicin, and tetracycline. Forty percent of the isolated CoNS resulted to
be multidrug-resistant and an extensively drug resistance profiles of observed only in a
strain of S. xylosus. This result is of great interest, since CoNS are known to be reservoirs of
resistance genes; that can easily be transmitted to other more virulent staphylococci like S.
aureus and Staphylococcus pseudintermedius (S. pseudintermedius), decreasing the chances of
success of therapeutic treatments [
9
]. Furthermore, the prevalence of MDR CoNS strains
found here is not only in accordance with data reported for CoNS isolated from wild boars
of other European countries [
10
,
11
], but also in CoNS strains isolated from pets suffering
from skin infections [16].
Referring to enterococci, the prevalence of isolation from the collected nasal swabs
was of 38%. This is an intriguing result, since it is generally unusual recover enterococci
from nasal samples of animals, which are reported here for the first time in wild boars.
Enterococci are known to be part of the gastrointestinal flora of humans and animals, but
being also opportunistic pathogens, they have been recognized, particularly E. faecalis and
E. faecium, as a significant cause of nosocomial infections [
43
]. E. faecalis was the most
predominant enterococcal species (93.8%; 15/16 strains) isolated from the here analyzed
wild boar nasal swabs. This obtained interesting result can be linked to wild boar rooting
activity and to the high presence of E. faecalis in the environment, since it is considered a
Animals 2022,12, 85 10 of 12
fecal indicator organism, which can be found and transmitted by different environmental
matrices (e.g., water, plants, soil, sediments, and sand). Thus, wildlife animals such as wild
boars, birds, and deer can be sources of enterococci in urban and rural environments, either
through direct deposition or in runoff [
43
]. In addition, the isolated
Enterococcus spp.
strains
displayed relevant resistance profiles. Resistance to penicillin was found in almost all of
the analyzed samples (93.7%) and 75% of the isolates were also resistant to ampicillin and
amoxicillin-clavulanic acid, followed by ciprofloxacin (68.7%). Similar resistance profiles
were described also by Olivera de Araujo et al. [
44
] for E. faecalis and other
Enterococcus spp.
strains isolated from fecal samples of wild Pampas foxes and Geoffroy’s cats in the Brazilian
Pampa biome. Furthermore, 43.7% of the isolated strains all identified as
E. faecalis
were
resistant to vancomycin. As reported by Zaheer et al. [
45
] which studied the antibiotypes
of E. faecalis isolated from humans, cattle, and environmental sources with a One Health
approach, also our VRE E. faecalis were multi-resistant to
β
-lactams, macrolides, aminogly-
cosides, fluoroquinolones, and tetracyclines. The most remarkable result emerging in this
study is that MDR and XDR strains isolated from nasal wild boar swabs were 62.5% and
25%, respectively. Indeed, it is known that enterococci can acquire resistance to numerous
antimicrobial agents, acting also as efficient donor of resistance genes.
The findings of the present study highlighted the isolation of CoNS and Enterococcus
strains showing worrying antibiotic resistance profiles in wild boars hunted in Campania
Region, southern Italy. The presence of MDR and XDR strains in nasal sample underlines
how these animals may contribute to the spread of resistant strains and their transmission
to other animals or even to humans. Furthermore, the occurrence of multi-resistance
in non-clinical strains may be considered a virulence characteristic that bacteria use to
improve their survival, proliferation, and their ability to colonize hosts. Therefore, a
continuous monitoring of the occurrence of these opportunistic bacteria in wildlife is
desirable. In addition, a constant surveillance of wild boars would be worthwhile, to
assess their role as reservoirs of antibiotic resistant bacteria and as sentinels of a possible
environmental contamination.
5. Conclusions
The present study reveals high colonization and antibiotic resistance in staphylococcal
and enterococcal isolates from nasal cavities of wild boars. Furthermore, nasal colonization
of multi-resistant CoNS and enterococci in these wild animals is alarming. A positive aspect
of these results was the absence of coagulase positive staphylococci, as methicillin-resistant
S. aureus, but this could be linked to the limitation of this study, represented by the low
number of collected samples. So, continuous monitoring and further surveillance studies
are required to better understand the emergence and spread of bacterial strains resistant to
antibiotics in wild ecosystems. The wild boar represents an important sentinel animal and
the bacteria of the two genera, Staphylococcus and Enterococcus, may be considered relevant
bacterial indicators of antibiotic resistance, a global public health concern.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/ani12010085/s1, Table S1: Matrix assisted laser desorption/ionization-time of flight mass
spectrometry (MALDI-TOF-MS) identification of Gram-positive bacteria isolated on Mannitol Salt
Agar (MSA).
Author Contributions:
Conceptualization, F.P.N. and L.D.M.; methodology, F.P.N. and L.D.M.;
software, F.P.N.; validation, F.P.N., F.F. and L.D.M.; investigation, F.P.N., G.F., E.S. and M.A.; data
curation, F.P.N. and L.D.M.; writing—original draft preparation, F.P.N. and L.D.M.; writing—review
and editing, F.P.N., F.F. and L.D.M. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
Not applicable. This study did not involve the use of living
wild boars, thus ethical approval was not required.
Animals 2022,12, 85 11 of 12
Informed Consent Statement:
Informed consent to participate in this research study was obtained
by licensed and specialized hunters, who provided wild boar nasal swabs.
Acknowledgments:
Authors wish to thank the licensed and specialized hunters (Antonio Parisi,
Gerardo D’Aponte, Antonino Abbate, and Giuseppe D’Amato) for providing nasal samples from
wild boars.
Conflicts of Interest: The authors declare no conflict of interest.
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