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MICROBIOLOGY
Food Microbiology 25 (2008) 196–201
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Short communication
Antibiotic resistance genes and identification of staphylococci collected
from the production chain of swine meat commodities
Desj Simeonia, Lucia Rizzottia, Piersandro Cocconcellib,c, Simona Gazzolab,c,
Franco Dellaglioa, Sandra Torriania,
a
Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy
b
Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29100 Piacenza, Italy
c
Centro Ricerche Biotecnologiche, Università Cattolica del Sacro Cuore, Via Milano 26, 26100 Cremona, Italy
Received 29 November 2006; received in revised form 20 March 2007; accepted 6 September 2007
Available online 15 September 2007
Abstract
Staphylococci harbouring antibiotic resistance (AR) genes may represent a hazard for human health and, as other resistant foodrelated bacteria, they contribute to the spread of AR. In this study, we isolated resistant staphylococci from an entire swine production
chain and investigated the occurrence of 11 genes [aac(60 )Ie-aph(200 )Ia, blaZ, mecA, vanA, vanB, ermA, ermB, ermC, tet(M), tet(O) and
tet(K)] encoding resistance to some antibiotics largely used in clinical practice. The 66 resistant staphylococcal isolates were identified
as Staphylococcus epidermidis (27 isolates), Staphylococcus aureus (12), Staphylococcus xylosus (12), Staphylococcus simulans (5),
Staphylococcus pasteuri (4), Staphylococcus carnosus (3), Staphylococcus lentus (2) and Staphylococcus sciuri (1). Specific-PCR detection
of AR genes showed the prevalence of the tet(K) gene in most of the isolates (89.4%), followed by tet(M) and ermC (about 75%); mecA
was detected in more than half of S. aureus and S. epidermidis isolates. The genes vanA and vanB were not retrieved. It was found that a
high proportion of coagulase-positive and -negative isolates are multidrug-resistant and some of them carry up to six AR genes. Our
findings show that the swine production chain is a source of antibiotic-resistant staphylococci suggesting the importance of resistance
surveillance in the food production environment.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Antibiotic resistance genes; PCR detection; Food chain; Staphylococcus; Multidrug resistance
1. Introduction
Staphylococci are ubiquitous Gram-positive bacteria
that represent part of the normal bacterial microflora of
the skin and mucosal surfaces of humans and animals.
Some staphylococcal species, such as Staphylococcus
aureus, Staphylococcus epidermidis and Staphylococcus
saprophyticus, are well known for their implications in
human health diseases (Yugueros et al., 2000). On the other
hand, some species of coagulase-negative staphylococci
(CNS) are considered technologically important in the
manufacturing processes of various meat-derived products,
especially dry fermented sausages, where they are used as
Corresponding author. Tel.: +39 045 802 7921; fax: +39 045 802 7051.
E-mail address: [email protected] (S. Torriani).
0740-0020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2007.09.004
starters to ensure the quality and safety of the final product
(Aymerich et al., 2003).
In the last decades, the spread of antibiotic resistance (AR)
in bacteria, including staphylococci, is increasing and may
represent a hazard for human health. Among antibioticresistant staphylococci, multidrug-resistant S. aureus strains
are of great public concern since resistances make more
difficult the treatment of infections. Moreover, a number of
CNS, such as several S. epidermidis strains, are important
hospital-acquired infection agents and the 80–90% of these
isolates are methicillin-resistant (de Mattos et al., 2003).
Transfer of antibiotic-resistant bacteria to humans (or
their AR genes to pathogens) via the food chain has
already been reported (Angulo et al., 2004; Phillips et al.,
2004). Some studies have evidenced the spreading of
resistant bacteria, such as staphylococci, enterococci and
lactic acid bacteria, during meat processing (Gevers et al.,
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D. Simeoni et al. / Food Microbiology 25 (2008) 196–201
2003; Huys et al., 2005; Rizzotti et al., 2005) and also the
transfer of AR determinants in natural microenvironments
between bacteria of different origins (Cocconcelli et al.,
2003). Thus, food-related bacteria, including the components of starters, have the potential to serve as a reservoir
of AR genes with the hazard of transferring these
determinants to other commensal or pathogenic species.
Up to now, many researches have focused on the spread
of resistant S. aureus in clinical environments (Marchese
et al., 2000; Roberts et al., 2000; da Silva Coimbra et al.,
2003), whereas a restricted number of investigations, or
Antimicrobial Resistance Research Programs (e.g. DANMAP), have regarded the presence in food of antibioticresistant S. aureus (Huys et al., 2005) and resistant CNS
(Perreten et al., 1998). The aim of this study was to
evaluate the occurrence of staphylococci resistant to
various antimicrobial agents in the entire production chain
of swine meat commodities, from animal farming to final
foods. Resistant staphylococcal isolates were collected and
examined for the presence of 11 AR genes by means of
specific PCRs; species identification and genetic correlation
among the isolates were determined, as well.
2. Materials and methods
2.1. Isolation of antibiotic-resistant staphylococci
Staphylococci were isolated from samples obtained from
various steps of the production chain of two factories
producing swine meat commodities located in the North of
Italy. The analysed samples included: eight faecal specimens collected from individual pigs; eight dry feedstuffs; 14
raw and processed pork meat products (raw unprocessed
pork products, carcasses, raw minced pork, fresh sausages)
after processing in the slaughterhouses, and six dry
fermented sausages ripened at the same factories.
Each sample was suspended in sterile 0.9% NaCl, then
an aliquot of this suspension was added to Trypticase Soy
Broth (Oxoid Italia, Milan, Italy) plus 4% NaCl and
incubated overnight at 37 1C. Aliquots of this culture were
streaked on selective plates containing Mannitol Salt Agar
(MSA, Oxoid) supplemented with one of the following
antibiotics: erythromycin (final concentration 16 mg ml1),
gentamycin (32 mg ml1), oxacillin (4 mg ml1), penicillin
G (0.5 mg ml1), tetracycline (32 mg ml1), vancomycin
(4 mg ml1), according to the breakpoint values defined by
NCCLS (2004). After incubation (37 1C; 24 h), representative colonies were picked and subcultured in tubes
containing Brain Heart Infusion (BHI; Oxoid) added with
the antimicrobial agent at the same concentration used for
isolation. The antibiotic-resistant isolates were purified and
maintained at 80 1C in 25% (v/v) glycerol.
2.2. Bacterial strains and DNA extraction
The staphylococcal isolates were deposited in the
laboratory culture collection. S. aureus subsp. aureus
197
DSM 20231T, S. epidermidis DSM 20044T, Staphylococcus
carnosus subsp. carnosus DSM 20501T and Staphylococcus
xylosus DSM 20266T were used as reference strains. Total
genomic DNA was extracted and purified from 2-ml
overnight cultures as described by Marmur (1961).
2.3. Identification and typing of staphylococci
Selected presumptive staphylococci were identified by
internal transcribed spacer-PCR (ITS-PCR) as described
by Jensen et al. (1993) and following the amplification
conditions of Mendoza et al. (1998). The amplicons were
resolved by agarose (3%, w/v) gel and each profile was
visually compared with those obtained from the staphylococcal reference strains. The identification of isolates
was then confirmed either by species-specific PCRs for
S. aureus, S. epidermidis, S. carnosus and S. xylosus
(Martineau et al., 1996, 1998; Aymerich et al., 2003) or
by 16S rRNA gene partial sequencing. A portion of the 16S
rRNA gene was amplified by PCR as previously described
(Rizzotti et al., 2005). The PCR products were purified by
using the Wizard SV Gel and PCR Clean-Up system
according to the package insert (Promega Corporation,
Madison, WI) and sequenced at Biomolecular Research
(BMR) Centre, University of Padova, Italy. The BlastN
program software was used for nucleotide sequence
analysis.
The RAPD typing method was carried out with the
primer D8635 on DNA extracted from the isolates as
described by Akopyanz et al. (1992). The patterns were
analysed using Gel Compar 4.0 software (Applied Math,
Kortrijk, Belgium).
2.4. PCR detection of antimicrobial resistance genes
The presence of genes involved in resistance to aminoglycosides [aac(60 )Ie-aph(200 )Ia gene], beta-lactams (blaZ,
mecA), glycopeptides (vanA, vanB), macrolide-lincosamidestreptogramins (ermA, ermB, ermC) and tetracyclines
[tet(M), tet(O), tet(K)] was determined in the isolates by
PCR amplification using the specific primers and the
conditions reported by Rizzotti et al. (2005).
2.5. Determination of phenotypic antimicrobial resistance
The resistances of staphylococci to specific antibiotics
were checked streaking each isolate on MSA plates added
with the antimicrobial agents at the concentrations
reported above. Minimum inhibitory concentrations
(MICs) were also determined for some antimicrobial agents
by the agar dilution method (NCCLS, 2004) on Isosensitest agar plates (Oxoid) added with 10% BHI.
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198
isolated from all kinds of analysed matrices except dry
fermented sausages. All the S. aureus isolates were found
only in fresh sausages, these meat products are generally at
low risk, with respect to consumer health, if properly
cooked. On the other hand, S. xylosus was the unique
species that was isolated from dry fermented sausages,
probably because of its better adaptation to the environmental conditions prevailing in these commodities.
3. Results and discussion
3.1. Isolation and identification of resistant staphylococci
Growth of antibiotic-resistant staphylococci was observed for 22 out of 36 food and food-related matrices and
a total of 66 resistant staphylococcal isolates were
considered for a deeper investigation on their taxonomic
position and AR pattern.
The combination of different genetic approaches was
applied to accurately identify the isolates. A preliminary
identification at the species level was obtained with the
ITS-PCR assay. However, very similar 16S–23S rDNA ITS
patterns can be displayed by diverse staphylococcal species
(Blaiotta et al., 2003). Consequently, species-specific PCRs
and/or 16S rRNA partial sequencing were applied to
confirm the identification.
The occurrence and distribution of Staphylococcus
species among the resistant isolates are shown in Table 1.
S. epidermidis was the predominant species and was
3.2. Prevalence of AR genes among resistant staphylococcal
isolates
Each staphylococcal isolate was tested for the presence
of the 11 AR genes. The PCR amplification results are
summarised in Table 2.
All except for six staphylococcal isolates held more than
one AR gene and a high proportion of these staphylococci
(72.7%) carried almost four different AR genes. In
particular, three S. epidermidis isolates carried six AR
genes that confer resistance to five different antibiotics.
Table 1
Identification and origin of the antibiotic-resistant staphylococcal isolates
Species
No. of isolates (%)
No. of isolates from isolation matrix
Faeces
S.
S.
S.
S.
S.
S.
S.
S.
epidermidis
aureus
xylosus
simulans
pasteuri
carnosus
lentus
sciuri
27
12
12
5
4
3
2
1
Total
(40.9)
(18.2)
(18.2)
(7.6)
(6.1)
(4.6)
(3.0)
(1.5)
Feedstuff
9
–
–
5
–
–
–
–
66
Raw meat
Processed meat
Dry fermented
sausage
2
1
11
–
4
–
–
3
–
–
4
12
–
–
4
–
–
–
–
–
3
–
–
–
–
–
11
18
20
3
3
–
5
–
–
–
14
–: Not detected.
Table 2
Incidence of AR genes in the antibiotic-resistant staphylococcal isolates
Species
S.
S.
S.
S.
S.
S.
S.
S.
epidermidis
aureus
xylosus
simulans
pasteuri
carnosus
lentus
sciuri
AR incidence
(%)
No. of
isolates
27
12
12
5
4
3
2
1
No. of positive isolatesa
tet(M)
tet(O)
tet(K)
17
12
6
5
4
2
2
1
1
–
–
–
–
–
–
–
25
11
8
5
4
3
2
1
74.2
1.5
89.4
–: Not detected.
a
vanA, vanB genes were not detected in any of the isolates.
b
Shortening of aac(60 )Ie-aph(200 )Ia.
ermA
ermB
ermC
aac6aph2b
blaZ
mecA
–
–
–
–
19
4
10
2
2
–
–
–
26
7
5
5
2
2
2
1
7
–
–
–
–
–
–
–
21
12
4
2
3
–
–
–
12
7
1
–
2
1
–
1
10.6
56.1
75.8
10.6
63.6
36.4
2
2
1
2
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These results confirm the large spread of multidrugresistant bacteria, including staphylococci, which can be
isolated from different environments, such as clinical,
animal and food samples (Santos Sanches et al., 2000;
Khan et al., 2005).
The fact that 72.7% of our isolates carried two
tetracycline resistance determinants reveals a great diffusion of this type of resistance. The carriage of multiple tet
genes was commonly found in individual Gram-positive
bacteria (Schwarz et al., 1998; Huys et al., 2005; Rizzotti
et al., 2005). The high incidence of tet(K) and tet(M)
genes in the isolated staphylococci, 89.4% and 74.2%,
respectively, can be explained by their usual genetic
locations. In fact, the presence of tet(K) gene on small
multicopy plasmids and tet(M) on conjugative transposons (Tn916–Tn1545 family) contributes to the spread
of these determinants (Chopra and Roberts, 2001).
Furthermore, we found tet(O) only in one S. epidermidis
strain; even Schwarz et al. (1998) observed tet(O) only
in few staphylococcal isolates and Schmitz et al. (2001)
did not retrieved this gene in tetracycline-resistant
S. aureus.
A large number (75.8%) of the resistant staphylococci
isolated here harboured ermC gene. This gene is frequently
located on small multicopy plasmids (Khan et al., 2002),
which are present in many different staphylococcal species.
The ermB gene is often carried by conjugative transposons
(Khan et al., 2002) and this could explain the diffusion of
ermB (56.1%) in the staphylococci isolated in this study.
Conversely, the ermA gene was detected in a lower number
of isolates.
The resistance to aminoglycosides [aac(60 )Ie-aph(200 )Ia
gene] was found only in seven isolates belonging to the
S. epidermidis species, mainly derived from animal faeces;
indeed this gene is usually more diffused in staphylococci
of human origin (Werckenthin et al., 2001).
The blaZ gene for beta-lactam resistance was detected in
63.6% of the staphylococci, including all S. aureus isolates
and 77.8% of the S. epidermidis isolates, indicating that
this determinant is widely spread among the CNS, too. The
resistance to methicillin (oxacillin), encoded by mecA gene,
was detected in 36.4% of the analysed staphylococci,
comprising 58.3% of the S. aureus isolates and 40.5% of
the isolated CNS (44.4% of S. epidermidis strains). The
mecA determinant may cause severe problems in humans
for the treatment of methicillin-resistant S. aureus (MRSA)
(Lee, 2003) and, in recent years, for S. epidermidis
infections, too (Kitao, 2003).
In this study, the vanA and vanB genes for vancomycin
resistance were not detected. Such antibiotic was the only
uniformly effective antimicrobial treatment for staphylococcal infections but several cases of vancomycin-resistant
staphylococci, including S. epidermidis with a low-level
(8–16 mg ml1) resistance, have been published (Krcmery
and Sefton, 2000). In our study, a S. epidermidis strain
showed a vancomycin MIC of 6 mg ml1. Probably, the
same mechanism of resistance retrieved in vancomycin-
199
intermediate S. aureus was involved in the reduced
susceptibility of this strain (Hanaki et al., 1998).
A study of the French National Monitoring Program
(RESABO) showed that CNS were less susceptible to
antimicrobial agents than coagulase-positive staphylococci
(Werckenthin et al., 2001). Conversely, our results showed
a comparable pattern of AR genes between CNS and
S. aureus isolates.
Since the plates used for isolation contained only one
antimicrobial agent, the resistance pattern of each staphylococcal isolate was established for the other antibiotics.
A close correspondence between resistance and PCR
results was found for erythromycin, gentamycin, penicillin
G and tetracycline. Instead, this correlation was not
observed for several isolates that carry mecA gene in the
presence of oxacillin as previously described by Martineau
et al. (2000) that reported the occurrence of S. aureus
strains with the mecA gene but susceptible to oxacillin.
3.3. Strain typing
To determine molecular epidemiological relatedness
among staphylococcal isolates belonging to the same
species, RAPD patterns were generated. The profiles
obtained from the S. aureus isolates are shown in Fig. 1.
Numerical analysis of the RAPD profiles revealed a
large genetic polymorphism among the staphylococcal
isolates, especially among S. aureus and S. epidermidis,
even if high similarity percentages were given by some of
them isolated from the same matrix (data not shown).
These data were combined with the results of the AR gene
screening showing the absence of clonal strains. As a
consequence no relevant contamination events have
Fig. 1. RAPD profiles obtained with the primer D8635 from S. aureus
isolates (lanes 1–12) and reference strain (lane 13). Lanes M: DNA
molecular size marker (1 kb plus DNA ladder, Invitrogen, Italy).
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occurred between different steps of the production chain
under consideration, even if a higher number of strains
should be analysed to confirm this.
In conclusion, this investigation provided evidences of
the wide occurrence of AR genes in resistant staphylococci
derived from various steps of the production chain of swine
meat commodities. It emerged that both coagulase-positive
and -negative resistant staphylococci carry a number of AR
genes demonstrating the spread of multidrug resistance in
bacteria of food origin too, and the importance of
resistance surveillance in the food production environment.
Acknowledgement
This study was supported by the ‘‘Fondazione Cassa di
Risparmio di Verona, Vicenza, Belluno e Ancona’’.
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