Abstract
Livestock-associated Staphylococcus aureus isolates, and especially those belonging to ST398, have been increasingly described in colonized and infected animals and humans, and also in food samples in several countries. The purpose of this study was to determine the frequency of S. aureus and methicillin-resistant S. aureus (MRSA) isolates in raw meat samples destined for food consumption in Tunisia, and to characterize the recovered isolates. One hundred sixty-nine food samples of animal origin were collected. Samples were inoculated onto selective mediums for S. aureus and MRSA recovery. Different molecular typing methods were implemented (pulsed-field gel electrophoresis [PFGE], multilocus sequence typing, spa-, agr-, and SCCmec typing). MRSA was detected in 2 of these 169 samples (1.2%), both of which were of chicken origin. The two MRSA isolates (one/sample) were typed as ST30-CC30-t012-agrIII-SCCmecV and ST398-CC398-t4358-agrI-SCCmecIVa. The MRSA ST398 strain presented resistance, in addition to β-lactams, to tetracycline (tet[M]) and erythromycin (erm[C]) and harbored the sen, hla, hlg, and hlgv virulence genes. Methicillin-susceptible S. aureus (MSSA) isolates were recovered from 42 of the 169 tested samples (24.8%). A high diversity of spa types (n=21) and SmaI-PFGE patterns (27 different profiles; 4 nontypeable) were detected among MSSA isolates. Four MSSA isolates were typed as ST398/CC398. The percentage of antimicrobial resistance and detected genes in MSSA isolates were as follows: tetracycline (28.6% tet[K] and tet[L]), kanamycin (9.5%, aph[3′]-IIIa), tobramycin (2.4%, ant[4′]-Ia), erythromycin (14.3%, erm[A], erm[C], msr[A]), and penicillin (95%). The genes lukS-lukF were detected in two MSSA isolates (4.5%), the gene tst in one isolate, and the gene eta in five isolates. To our knowledge, this is the first detection of MRSA and MSSA ST398 in food in an African country. The risk of transmission of S. aureus and MRSA carrying different antimicrobial resistance and virulence genes through the food chain cannot be ignored.
Introduction
S
Moreover, S. aureus is able to acquire multiple resistance genes, which limit the therapeutic options. Methicillin-resistant S. aureus (MRSA) isolates appeared in 1961, and in 1972, MRSA was isolated for the first time in animals (Devriese et al., 1972). Since then, they have been detected in different animal species (Madec and Haenni, 2010). In 2005, a new sequence type of MRSA (ST398) appeared in Europe associated with livestock animals (Armand-Lefevre et al., 2005). MRSA CC398 has been previously found in animals (especially in pigs), in people in contact with these animals (veterinarians and farmers) and their relatives (Khanna et al., 2008), and in food of animal origin (Lozano et al., 2009; Fessler et al., 2011). Until now, MRSA CC398 has been detected in European countries (Armand-Lefevre et al., 2005), Canada (Khanna et al., 2008), United States (Mediavilla et al., 2012), Asia (Lim et al., 2012), South America (Arriola et al., 2011), and Australia (Groves et al., 2013).
The studies carried out in Africa have been mainly focused on human isolates. Different CCs (CC1, CC5, CC15, CC30, CC88, CC152, CC121, or CC672) have been found among hospital and community MRSA and MSSA isolates, depending on the region (Breurec et al., 2011; Basset et al., 2015). Concerning data of Tunisia, one study carried out in healthy people only detected one MRSA belonging to CC80 (Ben Slama et al., 2011), and this clonal lineage was also observed in hospitalized children in this country (Ben Nejma et al., 2014). In some studies performed in healthy animals, the CCs identified among MRSA and MSSA isolates have also been diverse (CC1, CC6, CC22, CC72, CC130, CC80 or CC522 among others) (Gharsa et al., 2012a, 2012b), highlighting the predominance of CC133 among donkeys destined for food consumption (Gharsa et al., 2012a). Remarkably, only one study has identified the clonal lineage CC398 in one skin sample from a dog in Zambia, and this isolate was susceptible to methicillin (Youn et al., 2014).
The purpose of this study was to determine the frequency of S. aureus and MRSA isolates in raw meat samples destined for food consumption in Tunisia, and to investigate the content in antimicrobial resistance and virulence genes and the genetic lineages of recovered isolates.
Materials and Methods
Sampling and microbiological isolation
A total of 164 raw food samples of animal origin (84 poultry, 42 lambs, 30 calves, 4 horses, 3 rabbits, and 1 pig) were collected from 8 poultry markets, 6 butcheries, 3 farms, and 2 supermarkets in 10 different regions of Tunisia during October 2010 to March 2011. Samples were processed for recovery of MRSA and S. aureus as previously described (Lozano et al., 2012; Benito et al., 2014). One S. aureus–suspected colony per positive plate was selected and identified by conventional methods (Gram staining, catalase test, oxidase test, DNase production, and ability to coagulate rabbit plasma) (Biomerieux, Marcy l'Etoile, France). S. aureus identification was then confirmed by amplification of the species-specific nuc gene (Gómez-Sanz et al., 2010) (Supplementary Table S1; Supplementary Data are available online at
Antimicrobial susceptibility testing
Susceptibility to 18 antimicrobial agents was performed using the disk-diffusion method in accordance with the Clinical and Laboratory Standards Institute recommendations (CLSI, 2012). Antimicrobial agents tested were (charge in μg/disk): penicillin (10 units), oxacillin (1), cefoxitin (30), kanamycin (30), gentamicin (10), tobramycin (10), tetracycline (30), chloramphenicol (30), trimethoprim–sulfamethoxazole (1.25/23.75), erythromycin (15), clindamycin (2), amikacin (30), ciprofloxacin (5), mupirocin (5), vancomycin (30), teicoplanin (30), fusidic acid (10), and streptomycin (10). Double-disk diffusion test (D-test) with erythromycin and clindamycin disks was implemented in all isolates to detect inducible clindamycin resistance.
DNA extraction method
DNA of all S. aureus isolates recovered was extracted. For that, bacteria were harvested from brain–heart infusion agar plates, suspended in 45 μL of sterile water with 5 μL of lysostaphin (3000 U/mL) and incubated at 37°C for 10 min. After that, 45 μL of sterile water, 5 μL of proteinase K solution (2 mg/mL), and 150 μL of Tris HCl (0.1 M pH 8) were added. Cell suspensions were then incubated at 60°C for 10 min and later they were placed at 100°C for 5 min.
Detection of methicillin resistance genes and SCCmec-typing
The presence of the mecA gene was studied by polymerase chain reaction (PCR) in all oxacillin- and/or cefoxitin-resistant isolates using primers and conditions as previously reported (Gómez-Sanz et al., 2010). The mecC gene was also tested by PCR in all isolates (García-Álvarez et al., 2011). The SCCmec-typing was performed for mecA positive isolates by a multiplex PCR strategy described by Kondo et al. (2007) (Supplementary Table S1).
Detection of other antimicrobial resistance genes
The ribosomal methylases encoded by erm(A), erm(B), erm(C), and erm(T) genes, which confer resistance to erythromycin and clindamycin, and the efflux pump encoded by msr(A) gene, conferring resistance to erythromycin, were studied by PCR in erythromycin-resistant isolates. In addition, tet(K), tet(M), and tet(L) genes conferring resistance to tetracycline, and aph(3′)-IIIa and ant(4′)-Ia genes to kanamycin and tobramycin, were studied by PCR in all antimicrobial-resistant S. aureus isolates. We used the primers and conditions as mentioned by Lozano et al. (2009, 2012) (Supplementary Table S1).
Pulsed-field gel electrophoresis
All S. aureus isolates were characterized by pulsed-field gel electrophoresis (PFGE) with SmaI restriction enzyme digestion as previously described (Gómez-Sanz et al., 2010). The DNA fingerprints generated by PFGE were analyzed with the visual method and by the digitalized method by GelCompar II version 6.5 software (Applied Maths, Kortrijk, Belgium) generating a dendrogram according to Dice coefficient and unweighted-pair group method with arithmetic mean algorithm. In the strains in which no PFGE pattern was obtained with SmaI enzyme, the enzyme ApaI was used.
Other molecular typing methods of S. aureus isolates
Spa typing was performed in all S. aureus isolates as described elsewhere (Lozano et al., 2011). Identification of agr allele group (I–IV) was determined by multiplex PCR as described earlier (Shopsin et al., 2003). Multilocus sequence typing was performed in selected S. aureus isolates (isolates with spa types associated with ST398 and isolates with the two most common spa-types found in this study) (
Detection of staphylococcal toxin genes
All isolates were tested by PCR for the presence of sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, seu, tst, lukS-lukF, lukE-lukD, lukM, eta, etb, hla, hlb, hld, hlg, and hlgv genes (Lozano et al., 2011) (Supplementary Table S1).
Analysis of the possible origin of selected strains
The presence of several mobile genetic elements (MGEs) recently associated with an avian origin and the detection of the genes comprising the immune evasion cluster (IEC) (scn, chp, sak, sea, and/or sep) related to human origin were performed by PCR in ST398 strains (Van Wamel et al., 2006; Lowder et al., 2009) (Supplementary Table S1).
Results
Field survey results for S. aureus isolates
Forty-three food samples of the 164 studied (26.2%) contained S. aureus and 1 isolate per sample was further studied. Two of the S. aureus isolates were MRSA, and both were recovered from chicken samples and contained the mecA gene; the remaining 41 S. aureus isolates were methicillin susceptible (MSSA). The mecC gene was not identified in the isolates of this study.
Characteristics of MRSA isolates detected in this study
The two MRSA isolates showed different characteristics (Table 1). The spa-type of the isolate C4995 was t012, ST30, and agrIII. The mecA gene was carried by the SCCmecV. This isolate only presented resistance for β-lactams. It also contained the seg, sei, sem, seu, hla, and hld virulence genes. The second isolate, MRSA C5019, was ascribed to spa t4358, ST398, agrI, and SCCmecIVa. This strain showed a multiresistance phenotype that also included, in addition to β-lactams, tetracycline, erythromycin, and clindamycin (inducible type) and contained the resistance genes mecA, erm(C), and tet(M). Moreover, this strain harbored the genes sen, hla, hlg, and hlgv.
The two methicillin-resistant strains appear shaded.
The samples were obtained from 10 different regions of Tunisia: 1, Grand Tunis; 2, Menzel Bourguiba; 3, Bizerte; 4, Beja; 5, Mahdia; 6, Nabeul; 7, Mjez Beb; 8, Kef; 9, Sidi Bouzid; 10, Ben Guerden.
The Sequence Type (ST), after being determined by multilocus sequence typing, is indicated in parentheses.
The new spa type and the sequence type ST398 appear in bold.
PFGE patterns are indicated by A or S depending on the enzyme used (ApaI or SmaI, respectively).
New spa-type with a new repeat sequence (r652).
P, penicillin; OXA, oxacillin, FOX, cefoxitin; E, erythromycin; CC, clindamycin; TE, tetracycline; S, streptomycin; CIP, ciprofloxacin; K, kanamycin; TOB, tobramycin.
Characteristics of MSSA isolates detected in this study
The 41 MSSA isolated in our study showed 20 different spa types (Table 1). Four of the spa types were detected in 20 of the MSSA isolates (t005 in 10 isolates; t008 in 5 isolates; t189 in 3 isolates; and t024 in 2 isolates). Four of the MSSA isolates were ascribed to ST398 and to CC398, and they belonged to spa types t899 (two chicken isolates), t034 (one veal isolate), and a new spa type t13938, which presented a new repeat sequence (r652) (chicken isolate) (Table 1).
Characterization of the agr system showed a variable frequency with a predominance of agr group I (28 isolates, 68.3%), agr group II (10 isolates, 24.3%), and only 4 isolates belonged to agr group III (9.8%). The agr group IV has not been detected.
Characterization of antimicrobial resistance mechanisms and virulence genes among MSSA
All but 2 of the 41 MSSA isolates showed penicillin resistance (95%). Resistance to tetracycline was detected in 12 isolates (6 isolates containing tet[K]; 3 isolates of the combination tet[K] and tet[L]; 1 isolate tet[M]; and in 1 isolate, none of these genes was identified). The four isolates resistant to kanamycin harbored the aph(3′)-IIIa gene. One isolate was tobramycin resistant and contained the ant(4′)-Ia gene. The msrA, erm(A), erm(C), or erm(T) genes responsible for resistance to erythromycin were detected in four isolates.
All isolates carried at least two virulence genes. The genes lukS-lukF were detected in two isolates (4.9%), the gene tst was detected in one isolate, and the gene eta in four isolates.
PFGE patterns of S. aureus isolates
Analysis of SmaI macrorestriction profiles of the non-ST398 S. aureus isolates (37 MSSA and 1 MRSA) revealed 28 (S1–S27) different PFGE patterns. In pattern S2, two different subtypes were detected (S2a and S2b). Regarding the results of ApaI PFGE experiment performed in the five ST398 isolates (4 MSSA and 1 MRSA), two patterns were found (A1 and A2) and different subtypes of A1 were identified (A1a-A1c). The two MSSA ST398-t899 recovered from chicken samples showed the sample PFGE pattern (A1b) (Table 1).
Analysis of the possible origin of ST398 strains
None of the five ST398 strains studied harbored the MGEs associated with avian origin. Moreover, the genes of the IEC systems were not detected in two of these ST398 strains, but the remaining three ST398 strains (C5013, C5019, and C4993) presented the IEC type B (scn-chp-sak).
Discussion
The prevalence of S. aureus detected among the food samples analyzed in this study was 26.2%. The presence of this microorganism in meat has been studied in several countries (Schraft et al., 1992; Lozano et al., 2009; Huong et al., 2010; Benito et al., 2014). The percentage of food samples with S. aureus detected in our study was similar to that in some studies (Schraft et al., 1992; Huong et al., 2010). However, other studies detected higher prevalence rates (50–65%) (Kitai et al., 2004; Young et al., 2014), while others reported lower percentages (Normanno et al., 2007; Benito et al., 2014). Only 1.2% of our samples carried MRSA isolates. As occurred with S. aureus, the MRSA percentages detected in other studies have been variable (from 0 to 37%) (Normanno et al., 2007; Lozano et al., 2009; Fessler et al., 2011). These variations may be due in part to differences in the methodologies applied and especially to the origin of the samples. Regarding African studies, MRSA isolates have been previously detected in milk and meat samples in Nigeria, South Africa, and Kenya (Shitandi et al., 2004; Ateba et al., 2010; Ndahi et al., 2014). However, in those studies the mecA gene was not found or was not studied.
Although our MRSA prevalence was low, the risk of MRSA transmission through the food chain cannot be disregarded, and foodborne disease outbreaks caused by MRSA have been reported (Jones et al., 2002). One of our MRSA strains (C5019) was typed as ST398. To our knowledge, this is the first time that this clonal lineage has been reported in food samples in Africa. MRSA CC398 strains have been previously identified in food samples in other countries (Lozano et al., 2009), highlighting the study of Fessler et al. (2011) with a high prevalence of MRSA (37%) in poultry products (most of them of lineage CC398). Remarkably in our study, the MRSA ST398 isolate as well as three of the four MSSA ST398 isolates were obtained from chicken samples. We must take into consideration that in our study, only one sample from pig was included due to the fact that in this country, as in other Muslim countries, the consuming of pork is very low.
Our MRSA ST398 strain was typed as agrI-t4358-SCCmecIV, and exhibited a multiresistance phenotype. In addition to the MRSA ST398 strain (C5019), the other four MSSA strains belonged to the lineage ST398. Interestingly, two of them showed an identical PFGE pattern (A1b) and both were recovered from samples obtained in supermarkets of the same region. There are several recent publications in which it has been suggested that MRSA CC398 strains could have their origin in human MSSA CC398 strains (Price et al., 2012). Interestingly, the IEC type B was identified in two of the four MSSA ST398 strains as well as in the MRSA ST398 strain. The IEC system is a set of genes that allows evasion of the human defenses that are associated with strains of human origin. Its presence or absence in S. aureus strains isolated from food is very important, since it gives us information about the possible origin of the isolates (human or animal origin) (Benito et al., 2014). The presence of these genes in MSSA ST398 has been previously reported (Uhlemann et al., 2012). However, these genes are rarely found among MRSA ST398 strains (Haenni et al., 2011).
Our ST398 strains presented agrI and the spa types t034 (1 strain), t899 (two strains), t4358 (1 strain), and a new spa type t13938 (1 strain) with a new repeat sequence (r652) (Table 1). There are several spa types associated with lineage CC398, some of them being more commonly found in MRSA strains (t011, t108, t1197, or t1255 among others) and others in MSSA (t034 or t571) (Price et al., 2012). The spa type t4358 detected in the MRSA ST398 strain has been identified earlier in strains belonging to ST9, another clonal lineage also related to animals (Yan et al., 2014). The same occurs with spa type t899, which has been found in strains of both sequence types ST398 and ST9 by others (Larsen et al., 2012; Monaco et al., 2013). In fact, these two spa types (t4358 and t899) are the most common ones in pigs from China and Malaysia (Larsen et al., 2012).
The remaining MRSA strain (C4995) was typed as ST30-t012-agrIII-SCCmecV and did not carry the lukS-lukF genes encoding for PVL. However, PVL genes have been previously detected in MRSA strains belonging to ST30 (Hsu et al., 2006). Moreover, this toxin has been also found in MSSA CC30 isolates obtained from five major African towns (Breurec et al., 2011). MRSA CC30 is one of the most frequently found lineages in both colonization and causing infections in humans (van Belkum et al., 2009). Moreover, this clone has been also detected in samples from pigs (Agersø et al., 2012).
A high diversity of spa types was identified in the strains of this study. Twenty-three different spa types were identified, t005 and t008 being the most predominant ones. MSSA t005-CC22 strains have been previously detected in samples from different origins (Vincze et al., 2013; Aiken et al., 2014), and the spa type t008 is related to community strains and is one of the most common spa types identified among MSSA in human isolates (Tavares et al., 2014). Other spa types found in this study (t223, t267, t701, and t1166) were previously detected in healthy donkeys and sheep in Tunisia (Gharsa et al., 2012a, b). It is important to mention that the spa type t1166 is related to the ruminant-associated CC133.
Some relevant virulence factors were identified among our strains. The genes lukS-lukF were detected in two MSSA isolates (4.5%), the gene tst in one isolate, and the gene eta in five isolates. Some genes encoding enterotoxins, which are responsible for food poisoning, were also identified. Interestingly, many of our strains harbored genes that are part of the operon egc (seg, sei, sem, sen, and seo), but no strain showed the complete operon. The absence of one or more genes in the egc cluster has been previously reported (Lozano et al., 2011). In relation to the phenotype of resistance detected, most of the strains were penicillin resistant (95%), in accordance with other studies about MSSA strains (Lozano et al., 2011). It is important to note that 29.3% of the strains were tetracycline resistant. Tetracycline is widely used in the animal industry, and our strains were obtained from meat samples.
In conclusion, a relatively high (25%) and low (1.2%) percentage of MSSA and MRSA, respectively, were obtained among raw meat samples in our study. Among MSSA strains, a high diversity of spa types and PFGE patterns was obtained. Interestingly, to our knowledge, this is the first report of detection of MSSA and MRSA of the clonal lineage ST398 in food samples (meat of poultry and veal origins) in a country of the African continent. More information about the spread of S. aureus ST398 strains and other lineages in this continent is necessary. The risk of transmission of S. aureus and MRSA carrying different antimicrobial resistance and virulence genes through the food chain cannot be ignored, especially in raw meat.
Footnotes
Acknowledgments
This work was partially financed by project SAF2012-35474 from the Ministerio de Economía y Competitividad of Spain and Fondo Europeo de Desarrollo Regional. C. Lozano has a contract associated with project SAF2012-35474, and P. Gómez has a predoctoral fellowship from the Universidad de La Rioja (Spain).
Disclosure Statement
No competing financial interests exist.
References
Supplementary Material
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