Next Article in Journal
Agreement and Differences between Fat Estimation Formulas Using Kinanthropometry in a Physically Active Population
Next Article in Special Issue
The Effect of Ethanol Propolis Extracts on Inhibition of Growth of Fusarium solani on Hen Eggs
Previous Article in Journal
Intelligent Control of Mushroom Growing Conditions Using an Electronic System for Monitoring and Maintaining Environmental Parameters
Previous Article in Special Issue
Probiotics and Beneficial Microorganisms in Biopreservation of Plant-Based Foods and Beverages
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Diversity and Safety Aspects of Coagulase-Negative Staphylococci in Ventricina del Vastese Italian Dry Fermented Sausage

1
Dipartimento di Medicina e Scienze della Salute “V. Tiberio”, Università degli Studi del Molise, 86100 Campobasso, Italy
2
Istituto Zooprofilattico Sperimentale dell’Abruzzo e Molise (IZSAM), Sezione di Campobasso, 86100 Campobasso, Italy
3
Consiglio Nazionale delle Ricerche, Istituto di Scienze delle Produzioni Alimentari (CNR-ISPA), 73100 Lecce, Italy
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(24), 13042; https://doi.org/10.3390/app122413042
Submission received: 20 November 2022 / Revised: 3 December 2022 / Accepted: 16 December 2022 / Published: 19 December 2022
(This article belongs to the Special Issue State-of-Art of Microbial Concerns in Food Safety)

Abstract

:
Ventricina del Vastese is a traditional dry fermented sausage from Central Italy not yet characterized for the occurrence, identity and safety of coagulase-negative staphylococci (CNS), a bacterial group technologically important for this kind of product. Therefore, in this study, 98 CNS isolates from four manufacturers were differentiated using repetitive element palindromic PCR (Rep-PCR) and identified using 16S rRNA gene sequencing. These were examined for genes encoding biogenic amine (BA) production, resistance to aminoglycosides, β-lactams, tetracyclines and staphylococcal enterotoxins (SEs). Staphylococcus succinus (55%) predominated, followed by S. xylosus (30%), S. epidermidis (7.4%), S. equorum (3.1%), S. saprophyticus (3.1%) and S. warneri (1%). One S. succinus subsp. casei isolate was slightly β-hemolytic. SEs and the histidine decarboxylase gene hdcA were not detected, whereas the tyrosine decarboxylase gene tdcA was detected in four S. xylosus isolates. The blaZ beta-lactamase gene in an S. equorum isolate, tetracycline resistance genes tetK in six S. succinus isolates and tetA in one S. succinus isolate also bearing tetK were found. The product examined is characterized by a peculiar CNS species ratio and a low occurrence and diversity of AR transferable genes than found in other studies, as a probable consequence of production only with meat from animals raised in small farms with extensive rearing systems in which antibiotic usage is infrequent.

1. Introduction

Dry fermented sausages are produced from raw meat which is fermented and ripened by a composite microbiota. Microbial groups with the most relevant roles in the ripening process are lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS), which concur in making these products safe and tasteful. The main role of LAB is product acidification to different extents, according to the technological process, with inhibition of the most pathogenic and deteriorating microorganisms, whereas the most relevant role of CNS favors the development of an optimal red color through the formation of nitrosomyoglobin after stepwise nitrate reduction to nitric oxide and its combination with myoglobin. Both microbial groups are involved in the formation of flavors and aromatic substances [1].
The traditional versions of dry fermented sausages are manufactured according to ancient regional processes and exploit a naturally occurring microbiota. Among these, Ventricina del Vastese is a fermented dry sausage listed among traditional products by the Italian Ministry of Agriculture [2] and typical of a territory close to the Adriatic Coast, including parts of the Abruzzo and Molise Italian regions. Its recipe dates back to the 18th century, and its peculiarities are that it is made of knife-cut cubes of pork meat of about 2–4 cm sides, mixed with 20–30% of fat (w/w), which are salted and abundantly spiced with sweet and hot chili pepper powder (15–30 g/kg) and fill in natural casings. These casings are usually pig bladder or cecum and confer to the sausage a diameter of 9–20 cm. The product is aged for 100–150 days at temperatures not exceeding 13 °C. According to the production specifications fixed by an official document with legal value as per the Italian traditional food safeguard system, the “Disciplinare di Produzione” is approved by the international Slow Food Association [3] and proprietary for manufacturers belonging to the association “Associazione di Promozione e Tutela della Ventricina del Vastese”. These producers are committed to using meats from animals, also of autochthonous swine races, raised outdoors or in pens with no less than five square meters of space per head. Animals are fed exclusively with cereals, legumes, fruits and acorns produced locally. Use of preservatives, including nitrate, is not allowed.
Ventricina del Vastese was little characterized for CNS species composition and identity, with just one study carried out for a single producer to compare the effects of ripening in natural conditions or in a ripening chamber, in which the safety of isolates was not evaluated [4].
Coagulase-negative staphylococci (CNS) are naturally associated with dry fermented sausages as they normally colonize human and animal skin [5]. This bacterial group includes strains which are able to develop the desired flavors and aroma compounds from proteolysis and lipolysis [1]. CNS starters for meat products are commercially available [6], but the safety of this bacteria must be ascertained at the strain level as risk characters, such as the presence of transferable antibiotic resistance (AR) genes and staphylococcal enterotoxins (SEs), were shown to occur rather frequently [7,8]. In addition, the sausage-associated CNS species Staphylococcus equorum and S. succinus were also isolated from human clinical specimens [5], so the safety characteristics of the individual strains naturally present or used as starter cultures in fermented meats must be carefully evaluated. As a consequence, CNS species are not included in the updated list of biological agents with a qualified presumption of safety (QPS) status by the European Food Safety Authority [9].
As for other traditional naturally fermented and ripened products, the characterization of the dominant microbiological consortia for Ventricina del Vastese sausage is also required to gain knowledge of the bacterial species and strains involved in product transformation and for a selection of the technologically best-suited strains, devoid of risk characters, to be used as autochthonous starter cultures for quality and safety improvement.
Therefore, in this study, the product was characterized with respect to the presence, identity and safety status of CNS by analyzing samples from four artisanal manufacturers adhering to the association “ Associazione di Promozione e Tutela della Ventricina del Vastese” who promoted this research to obtain an evaluation of the uniqueness, authenticity and safety of their product on a scientific basis.

2. Materials and Methods

2.1. Bacterial Strains and Culture Conditions

Bacterial strains used in this study were all new isolates from Ventricina del Vastese sausages. For their isolation, Ventricina del Vastese samples were collected in the period January–May 2021 at 0, 20, 50 and 150 days of ripening from four manufacturers in the production area who use meat from local farms that raise no more than 15 pigs at a time. Sausage samples of 10 g were homogenized in 90 mL of sterile physiological solution (NaCl 9 g/L). The homogenates were serially diluted and inoculated in plates of mannitol salt agar (MSA) medium (Biolife Italiana, Milan, Italy) and incubated aerobically at 37 °C for 48 h. Pure cultures of the isolates were obtained by double streaking single colonies from the count plates on the same medium. A single colony of each isolate was grown in brain heart infusion (BHI) broth (Biolife Italiana) in the above conditions prior to DNA extraction. Broth cultures from single colonies were stored at −80 °C in the same medium with 20% (v/v) glycerol added for long term maintenance.
The determination of hemolytic activity was carried out and interpreted as described by Zell et al. [10].

2.2. DNA Isolation

DNA was extracted from 1 mL of fresh culture using the genomic RBC Bioscience DNA extraction kit (Diatech Labline, Jesi, AN, Italy), according to the manufacturer instructions. The quantity and integrity of the extracted DNA were checked by comparison with known amounts of lambda DNA (ThermoFisher Scientific, Rodano, MI, Italy) on 1.5% w/v agarose gels in 1 × TAE buffer (80 mM Tris-acetate, 2 mM EDTA, pH 8.0) stained with 1:10,000 diluted GelRed (Biotium, Società Italiana Chimici, Rome, Italy) and run at 120 V.

2.3. PCR Assays

All of the PCR tests were carried out using the EmeraldAmp GT PCR Master Mix Takara Clontech (Diatech, Jesi, Italy). Repetitive element palindromic PCR (Rep-PCR) was carried out using the GTG5 primer, as described by Versalovich et al. [11]. Primers 27f/1492r [12] were used to amplify a 1494 bp region of the 16S rRNA gene. Screenings for tyrosine decarboxylase tdcA and histidine decarboxylase hdcA genes were carried out according to Lagioia et al. [13] and Rossi et al. [14], respectively. Primers used to detect the tetracycline efflux genes tetA/C, tetG and the ribosomal protection proteins for tetracycline resistance genes tetM, O, P, Q, S, T and W are those designed by Yu et al. [15]. Other antibiotic resistance genes, i.e., blaZ, mecA, tetK, tetL, aac(6’)-Ie+aph(2’), aph(3’)-IIIa, ermA, ermB, ermC and msrA, were sought, as referenced by Rebecchi et al. [8]. In addition, other primer pairs were designed in this study using search of AR genes found in staphylococci in the NCBI database (https://www.ncbi.nlm.nih.gov/nucleotide/ (accessed on 22 October 2022) and verification of correct annealing and specificity by Blastn (https://blast.ncbi.nlm.nih.gov/ (accessed on 22 October 2022). These primer pairs are aac6f (5’-CCTTGCGATGCTCTATG-3’)/aac6r (5’-TCCCCGCTTCCAAGAG-3’) and ant6f (5’-GCGCAAATATTAATATACCTAAA-3’)/ant6r (5’-GGGCAATAAGGTAAGATCA-3’), respectively, amplifying fragments of aac6 (204 bp) and ant6 (157 bp) families of aminoglycoside resistance encoding genes. SE genes were sought according to Omoe et al. [16].
As positive control bacterial strains were not available for the genes screened, the DNA suitability for amplification was constantly checked by running parallel PCR reactions for the 16S rRNA gene. PCR products were separated using electrophoresis, as described above. In addition, PCR tests were repeated three times to ensure the reliability of the results.

2.4. Sequencing

Sequencing of the amplification products was carried out on both strands by Eurofins Genomics on amplicons purified with the Wizard® SV Gel and PCR Clean-Up System (Promega Italia Srl, Milan, Italy) with primers 27f/1492r for 16S rRNA gene and the same primers used for amplification of the other genes sequenced. All genes detected in the screenings were sequenced for identity confirmation.

2.5. Data Analyses

Data of CNS counts for the different producers were compared using unweighted pair group method with arithmetic averages (UPGMA) and correlation similarity index using PAST 4.03 free statistical software downloaded from https://past.en.lo4d.com/windows (accessed on 22 October 2022). Rep-PCR profiles were compared using BioNumerics V5.10 software (Applied-Maths, Sint-Martens-Latem, Belgium), with the dice coefficient for pairwise comparison and UPGMA clustering.

3. Results and Discussion

3.1. CNS Microbiota Composition

The analysis of the Ventricina del Vastese samples for CNS content at different times resulted in the numerical trends shown in Figure 1A, where it is possible to observe that a similar evolution of this bacterial group took place in the products of the four manufacturers examined. Though for manufacturer B lower numbers were most often present, positive correlations were determined among the count data series (Figure 1B).
The maximum count data approached those reported for other dry sausage types from Southern Italy [17,18], but the persistence of the highest count values until day 50 can be a consequence of slow drying due to the large diameter of the product.
The isolates obtained from each manufacturer of Ventricina del Vastese are listed in Table 1 according to the time of isolation.
The number of isolates per manufacturer depended on the number of production lots analyzed, two for manufacturers A, C and D and one for manufacturer B, and on the number of colony morphologies observed, which ranged between two and seven.
The Rep-PCR genotypes of the isolates are shown in Figure 2, where they are clustered according to the similarity percentage and with species indication for the isolates identified by the sequencing of the 16S rRNA gene.
For clusters of isolates sharing more than a 90% profile similarity, only one component was sequenced. As it can be observed, the most numerous group was that of the S. succinus isolates, representing 55% of the total, whereas 30% of the isolates were assigned to the species S. xylosus, 7.4% to S. epidermidis, 3.1% to S. equorum and 3.1% to S. saprophyticus. Only one isolate was identified as S. warneri. This species association is typical of the Southern-European type of fermented dry sausages with low acidity levels in which 3-methyl-1-butanol, acetoin and diacetyl represent distinctive aroma compounds formed by S. succinus and S. xylosus [19].
All the species identified, including those mostly implicated in human infections, i.e., S. epidermidis [20] and S. saprophyticus [21], were detected at the end of ripening (Table 1, Figure 2). The S. epidermidis isolates were retrieved from manufacturers A and C, whereas S. saprophyticus was also isolated from manufacturer D. It can be mentioned that two isolates from manufacturer C grown on MSA, omitted from Figure 2, were identified as Bacillus velezensis, a bacterial species under consideration as a starter for fermented foods [22] whose role in fermented sausages should be examined.
In Ventricina del Vastese sausage, the species S. succinus predominated among CNS and was isolated from all manufacturers. The occurrence of S. succinus in dry-fermented sausages from Italy was previously reported, but its frequency of isolation was at most 14.7% in sausages from the Campania region [23]. In a screening of CNS species present in fermented sausages from different European countries, S. succinus was identified only in Belgian products as a minor component of the CNS population [24]. Therefore, in Ventricina del Vastese, this species is exceptionally predominating, possibly as a consequence of the manufacturing method that creates high salt concentrations on the surface of the meat cubes constituting the sausage. Indeed, this species can be isolated from fermented products with high salt content, such as doenjang, a traditional fermented soybean Korean food [25]. Strains of S. succinus are able to colonize the surfaces of the manufacturing plants, as found in a study aimed at the characterization of the CNS population in a small establishment producing French traditional dry fermented sausages [26].
The S. succinus strains from fermented soybean products did not show hazardous traits, as they were susceptible to all of the antibiotics tested, did not form biogenic amines and were not hemolytic [25]. Among them, the strain 14BME20 was selected as a starter candidate, and its genomic analysis confirmed that it is devoid of virulence factor-encoding genes and possesses genes for lipid degradation that can lead to the formation of volatile compounds [27]. In addition, in this study, the S. succinus isolates had a low frequency of AR determinants and did not bear genes for the production of the most dangerous BAs, histamine and tyramine [13,14]. The only BA formed by S. succinus is cadaverine, as a lysine decarboxylase-encoding gene was reported to be present in the isolates producing low levels of this compound [25].
In the S. succinus 14BME20 genome (Acc. N. NZ_CP018199.1), a nitric oxide synthase (NOS) gene is found, indicating the possible production of nitric oxide (NO) by this bacterial species from L-arginine in the presence of oxygen and NADPH [28], which could contribute to the meat’s red color, though S. succinus does not reduce nitrate with the exception of the subspecies S. succinus subsp. casei [29]. As the addition of nitrate is not allowed in Ventricina del Vastese, the NOS activity of S. succinus could compensate for the red color maintenance.
The S. succinus isolates exhibited a lower diversity compared to the other CNS species in Ventricina del Vastese, with some clusters joined at a similarity above 90% that comprised isolates from different manufacturers. This bacterial group could, therefore, represent a typical component of the CNS population in the production area to be characterized for its influence on the quality and distinctness of the product. Indeed, S. succinus was reported to produce species-specific volatile compounds when used as a starter culture [30].

3.2. Safety Assessment of Ventricina Del Vastese CNS

Among the CNS isolates examined, a minority were found to bear hazardous traits, with four strains of S. xylosus potentially able to form tyramine due to the presence of a tdcA gene and seven strains harboring AR determinants (Figure 2). The AR genes found in this study, blaZ and tetK, were reported to be frequent in CNS [7,8], whereas the mecA conferring methicillin resistance and the mrsA encoding a macrolide efflux protein previously found in some of the species identified in this study [7,8] were absent in the isolates from Ventricina del Vastese sausage. In addition, the gene blaZ, reported by Rebecchi et al. [8] to be the most prevalent in sausage-associated CNS, was infrequent in this study. As a strong correlation was found between the presence of blaZ and tetK and a phenotypic resistance to penicillin and tetracycline, respectively [8], the CNS isolates carrying these genes found in this study are likely phenotypically resistant as well.
Importantly, no multidrug resistance (MDR) genetic profiles were observed, with only one strain possessing two AR genes, tetK and tetA. The gene tetK is plasmid-encoded in S. aureus [31] and, therefore, is prone to be transferred. On the other hand, the gene tetA found in this study was not previously detected in CNS from food, and, through a database search, it was found to be chromosomally encoded in an S. cohnii clinical isolate (Acc. N. Accession: UHEC01000001.1) among CNS and in coagulase-positive staphylococci.
Notably, the AR gene-harboring isolates found in this study were mostly associated with a single manufacturer, with only one exception, suggesting a localized selection of AR CNS. Therefore, examining bacterial isolates for AR might be useful to identify trends of AR bacteria selection at a farm level.
Staphylococcal enterotoxins were absent in the isolates obtained in this study, which is different than the findings by Soares Nunes et al. [32] who isolated S. epidermidis, S. succinus, S. xylosus and S. saprophyticus strains which harbored most often seb/sec and sea, followed by sed/seh/selm and sei/seln and were also expressed in vitro. Finally, weak hemolytic activity was only found in the isolate S. succinus subsp. casei A27.
The low frequency of the AR genes in CNS from Ventricina del Vastese sausage and the absence of MDR are indicative of a production process in which selective pressure for AR is weak, possibly because of the little need for antibiotic usage in animals raised in small farms with extensive rearing systems. Indeed, Fontana et al. [33] observed that extensive farming for artisanal sausage production determines a low occurrence of AR genes in CNS compared to production on an industrial scale with animals farmed intensively. However, even a low level of AR gene occurrence in CNS from Ventricina del Vastese indicates the need to monitor the spread of these genes and adopt measures that reduce their presence.
The effectiveness of autochthonous starter cultures including CNC in improving the safety of fermented sausages was demonstrated by the reduction in BA formation [34]. Therefore, the isolation and characterization of CNS present in Ventricina del Vastese, as well as in other similar products, are indispensable to allow the selection of strains to be tested as autochthonous starters eventually able to reduce BA formation and the occurrence of strains with risk factors such as transferable AR genes.

4. Conclusions

The characterization of the CNS microbiota of Ventricina del Vastese sausage highlighted particular aspects of this product, common to four producers, i.e. the predominance of the species S. succinus which comprises microorganisms of proven capacity to form aroma compounds; the low occurrence of species of dubious safety status, such as S. epidermidis and S. saprophyticus; and the absence of isolates harboring SEs. However, the occurrence of the S. xylosus strains with the tyrosine decarboxylase gene tdcA, isolates bearing the β-lactamase gene blaZ and the tetracycline efflux pumps tetK and tetA indicated that an improvement in the safety status of the product with respect to the occurrence of hazardous genetic traits is necessary.
Defining the specific CNS microbiota of Ventricina del Vastese sausage and the individual safety status of isolates is a first step toward the exploitation of autochthonous bacterial strains to supplant those with hazardous traits that naturally occur. The evaluation of single strains for their potential use as starter cultures, with the participation of producers, should follow. Indeed, further investigations are needed, including the full genetic characterization of the isolates using whole genome sequencing and the definition of their technological properties in production trials.

Author Contributions

Conceptualization, G.C. and P.P.; methodology, C.A.; software, F.R.; validation, F.R. and P.P.; formal analysis, C.A.; investigation, C.A. and L.M.; resources, G.C.; data curation, P.P.; writing—original draft preparation, C.A.; writing—review and editing, F.R.; supervision, G.C. and L.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.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data supporting reported results can be provided by the authors upon request.

Acknowledgments

Ventricina del Vastese sausage manufacturers are acknowledged for providing samples to carry out this research.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Palavecino Prpich, N.Z.; Camprubí, G.E.; Cayré, M.E.; Castro, M.P. Indigenous Microbiota to Leverage Traditional Dry Sausage Production. Int. J. Food Sci. 2021, 2021, 6696856. [Google Scholar] [CrossRef] [PubMed]
  2. Italian Ministry of Agriculture. Ventiduesima Revisione Dell’elenco Dei Prodotti Agroalimentari Tradizionali. Available online: https://www.politicheagricole.it/flex/cm/pages/ServeBLOB.php/L/IT/IDPagina/17979 (accessed on 22 October 2022).
  3. Fondazione Slow Food per la Biodiversità Onlus. Ventricina del Vastese Presidio Slow Food. Available online: https://www.fondazioneslowfood.com/it/presidi-slow-food/ventricina-del-vastese/ (accessed on 22 October 2022).
  4. Amadoro, C.; Rossi, F.; Piccirilli, M.; Berardino, L.; Colavita, G. Studio della flora microbica pro-tecnologica nella ventricina. Ing. Aliment. 2013, 50, 51–54. [Google Scholar]
  5. Nováková, D.; Sedláček, I.; Pantůček, R.; Štětina, V.; Švec, P.; Petráš, P. Staphylococcus equorum and Staphylococcus succinus isolated from human clinical specimens. J. Med. Microbiol. 2006, 55, 523–528. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Van Ba, H.; Seo, H.W.; Kim, J.H.; Cho, S.H.; Kim, Y.S.; Ham, J.S.; Park, B.Y.; Kim, H.W.; Kim, T.B.; Seong, P.N. The effects of starter culture types on the technological quality, lipid oxidation and biogenic amines in fermented sausages. LWT 2016, 74, 191–198. [Google Scholar] [CrossRef]
  7. Marty, E.; Bodenmann, C.; Buchs, J.; Hadorn, R.; Eugster-Meier, E.; Lacroix, C.; Meile, L. Prevalence of antibiotic resistance in coagulase-negative staphylococci from spontaneously fermented meat products and safety assessment for new starters. Int. J. Food Microbiol. 2012, 159, 74–83. [Google Scholar] [CrossRef] [PubMed]
  8. Rebecchi, A.; Miragoli, F.; Lopez, C.; Bassi, D.; Fontana, C. Exploring Coagulase-Negative Staphylococci Diversity from Artisanal Llama Sausages: Assessment of Technological and Safety Traits. Microorganisms 2020, 8, 629. [Google Scholar] [CrossRef] [PubMed]
  9. EFSA BIOHAZ Panel. Updated List of QPS-Recommended Biological Agents for Safety Risk Assessments Carried Out by EFSA. Available online: https://zenodo.org/record/6902983#.Y3i0VX3MLIV (accessed on 22 October 2022).
  10. Zell, C.; Resch, M.; Rosenstein, R.; Albrecht, T.; Hertel, C.; Götz, F. Characterization of toxin production of coagulase-negative staphylococci isolated from food and starter cultures. Int. J. Food Microbiol. 2008, 127, 246–251. [Google Scholar] [CrossRef]
  11. Versalovic, J.; Schneider, M.; De Bruijn, F.J.; Lupski, J.R. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol. Cell. Biol. 1994, 5, 25–40. [Google Scholar]
  12. Fredriksson, N.J.; Hermansson, M.; Wilén, B.M. The choice of PCR primers has great impact on assessments of bacterial community diversity and dynamics in a wastewater treatment plant. PLoS ONE 2013, 8, e76431. [Google Scholar] [CrossRef]
  13. La Gioia, F.; Rizzotti, L.; Rossi, F.; Gardini, F.; Tabanelli, G.; Torriani, S. Identification of a tyrosine decarboxylase gene (tdcA) in Streptococcus thermophilus 1TT45 and analysis of its expression and tyramine production in milk. Appl. Environ. Microbiol. 2011, 77, 1140–1144. [Google Scholar] [CrossRef] [Green Version]
  14. Rossi, F.; Gardini, F.; Rizzotti, L.; La Gioia, F.; Tabanelli, G.; Torriani, S. Quantitative analysis of histidine decarboxylase gene (hdcA) transcription and histamine production by Streptococcus thermophilus PRI60 under conditions relevant to cheese making. Appl. Environ. Microbiol. 2011, 77, 2817–2822. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Yu, Z.; Michel, F.C., Jr.; Hansen, G.; Wittum, T.; Morrison, M. Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance. Appl. Environ. Microbiol. 2005, 71, 6926–6933. [Google Scholar] [CrossRef] [PubMed]
  16. Omoe, K.; Hu, D.L.; Takahashi-Omoe, H.; Nakane, A.; Shinagawa, K. Comprehensive analysis of classical and newly described staphylococcal superantigenic toxin genes in Staphylococcus aureus isolates. FEMS Microbiol. Lett. 2005, 246, 191–198. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Rossi, F.; Tofalo, R.; Torriani, S.; Suzzi, G. Identification by 16S-23S rDNA intergenic region amplification, genotypic and phenotypic clustering of Staphylococcus xylosus strains from dry sausages. J. Appl. Microbiol. 2001, 90, 365–371. [Google Scholar] [CrossRef] [PubMed]
  18. Mauriello, G.; Casaburi, A.; Blaiotta, G.; Villani, F. Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy. Meat Sci. 2004, 67, 149–158. [Google Scholar] [CrossRef]
  19. Ravyts, F.; Steen, L.; Goemaere, O.; Paelinck, H.; De Vuyst, L.; Leroy, F. The application of staphylococci with flavour-generating potential is affected by acidification in fermented dry sausages. Food Microbiol. 2010, 27, 945–954. [Google Scholar] [CrossRef]
  20. Heilmann, C.; Ziebuhr, W.; Becker, K. Are coagulase-negative staphylococci virulent? Clin. Microbiol. Infect. 2019, 25, 1071–1080. [Google Scholar] [CrossRef]
  21. Hur, J.; Lee, A.; Hong, J.; Jo, W.Y.; Cho, O.H.; Kim, S.; Bae, I.G. Staphylococcus saprophyticus Bacteremia originating from Urinary Tract Infections: A Case Report and Literature Review. Infect. Chemother. 2016, 48, 136–139. [Google Scholar] [CrossRef]
  22. Hong-Eun, N.; Sojeong, H.; Yoon-Su, K.; Tao, K.; Gawon, L.; Jong-Hoon, L.; Do-Won, J. The safety and technological properties of Bacillus velezensis DMB06 used as a starter candidate were evaluated by genome analysis. LWT 2022, 161, 113398. [Google Scholar]
  23. Milicevic, B.; Danilovic, B.; ZDolec, N.; Kozachinski, L.; Dobranic, V.; Savic, D. Microbiota of the fermented sausages: Influence to product quality and safety. Bulg. J. Agric. Sci. 2014, 20, 1061–1078. [Google Scholar]
  24. Van Reckem, E.; Charmpi, C.; Van der Veken, D.; Borremans, W.; De Vuyst, L.; Weckx, S.; Leroy, F. Application of a High-Throughput Amplicon Sequencing Method using a partial region of the tuf gene to Chart the Bacterial Communities that Are Associated with European Fermented Meats from Different Origins. Foods 2020, 9, 1247. [Google Scholar] [CrossRef] [PubMed]
  25. Jeong, D.W.; Lee, B.; Her, J.Y.; Lee, K.G.; Lee, J.H. Safety and technological characterization of coagulase-negative staphylococci isolates from traditional Korean fermented soybean foods for starter development. Int. J. Food Microbiol. 2016, 236, 9–16. [Google Scholar] [CrossRef] [PubMed]
  26. Corbière Morot-Bizot, S.; Leroy, S.; Talon, R. Staphylococcal community of a small unit manufacturing traditional dry fermented sausages. Int. J. Food Microbiol. 2006, 108, 210–217. [Google Scholar] [CrossRef] [PubMed]
  27. Jeong, D.W.; Lee, J.H. Complete Genome Sequence of Staphylococcus succinus 14BME20 Isolated from a Traditional Korean Fermented Soybean Food. Genome Announc. 2017, 5, e01731-16. [Google Scholar] [CrossRef] [PubMed]
  28. Chen, Y.; Rosazza, J.P.N. Purification and characterization of nitric oxide synthase (NOSnoc) from a Nocardia species. J. Bacteriol. 1995, 177, 5122–5128. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Place, R.B.; Hiestand, D.; Burri, S.; Teuber, M. Staphylococcus succinus subsp. casei subsp. nov., a dominant isolate from a surface ripened cheese. Syst. Appl. Microbiol. 2002, 25, 353–359. [Google Scholar]
  30. Jeong, D.W.; Lee, H.; Jeong, K.; Kim, C.T.; Shim, S.T.; Lee, J.H. Effects of starter candidates and NaCl on the production of volatile compounds during soybean fermentation. J. Microbiol. Biotechnol. 2019, 29, 191–199. [Google Scholar] [CrossRef]
  31. Guay, G.G.; Khan, S.A.; Rothstein, D.M. The tet(K) gene of plasmid pt181 of Staphylococcus aureus encodes an efflux protein that contains 14 transmembrane helices. Plasmid 1993, 30, 163–166. [Google Scholar] [CrossRef]
  32. Soares Casaes Nunes, R.; Mere Del Aguila, E.; Paschoalin, V.M. Safety Evaluation of the Coagulase-Negative Staphylococci Microbiota of Salami: Superantigenic Toxin Production and Antimicrobial Resistance. Biomed. Res. Int. 2015, 2015, 483548. [Google Scholar] [CrossRef] [Green Version]
  33. Fontana, C.; Patrone, V.; Lopez, C.M.; Morelli, L.; Rebecchi, A. Incidence of Tetracycline and Erythromycin Resistance in Meat-Associated Bacteria: Impact of Different Livestock Management Strategies. Microorganisms 2021, 9, 2111. [Google Scholar] [CrossRef]
  34. Dias, I.; Laranjo, M.; Potes, M.E.; Agulheiro-Santos, A.C.; Ricardo-Rodrigues, S.; Fialho, A.R.; Véstia, J.; Fraqueza, M.J.; Oliveira, M.; Elias, M. Co-Inoculation with Staphylococcus equorum and Lactobacillus sakei Reduces Vasoactive Biogenic Amines in Traditional Dry-Cured Sausages. Int. J. Environ. Res. Public Health 2021, 18, 7100. [Google Scholar] [CrossRef] [PubMed]
Figure 1. CNS evolution in Ventricina del Vastese sausage of four manufacturers A, B, C and D (A) and correlation among the evolution trends for the different manufacturers (B).
Figure 1. CNS evolution in Ventricina del Vastese sausage of four manufacturers A, B, C and D (A) and correlation among the evolution trends for the different manufacturers (B).
Applsci 12 13042 g001
Figure 2. Clustered Rep-PCR profiles of CNS isolated from Ventricina del Vastese sausage. Identification based on 16S rRNA gene is shown for isolates representing clusters separated above 90% similarity.
Figure 2. Clustered Rep-PCR profiles of CNS isolated from Ventricina del Vastese sausage. Identification based on 16S rRNA gene is shown for isolates representing clusters separated above 90% similarity.
Applsci 12 13042 g002
Table 1. CNS isolates from manufacturers A, B, C and D with time of isolation.
Table 1. CNS isolates from manufacturers A, B, C and D with time of isolation.
ManufacturerABCD
Day 0A1, A3, A4, A5, A6, A7, A8 B1, B2 C1, C2, C3, C4, C5, C6, C7, C8 D1, D2, D3, D4, D5, D6, D7
Day 20A9, A10, A11, A12, A14, A16, A17B4, B6, B8C9, C10, C11, C12, C13, C14, C15, C16, C17D8, D9, D10, D11, D12, D13, D14, D15, D16
Day 50A22, A23, A24, A26, A27, A28 B10, B11C18, C19, C20, C21, C22, C23, C24D17, D18, D19, D20, D21, D22, D23
Day 150A29, A31, A32, A33, A34B12, B15C25, C26, C27, C28, C29, C30D24, D25, D26, D27, D28, D29, D30
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Amadoro, C.; Rossi, F.; Poltronieri, P.; Marino, L.; Colavita, G. Diversity and Safety Aspects of Coagulase-Negative Staphylococci in Ventricina del Vastese Italian Dry Fermented Sausage. Appl. Sci. 2022, 12, 13042. https://doi.org/10.3390/app122413042

AMA Style

Amadoro C, Rossi F, Poltronieri P, Marino L, Colavita G. Diversity and Safety Aspects of Coagulase-Negative Staphylococci in Ventricina del Vastese Italian Dry Fermented Sausage. Applied Sciences. 2022; 12(24):13042. https://doi.org/10.3390/app122413042

Chicago/Turabian Style

Amadoro, Carmela, Franca Rossi, Palmiro Poltronieri, Lucio Marino, and Giampaolo Colavita. 2022. "Diversity and Safety Aspects of Coagulase-Negative Staphylococci in Ventricina del Vastese Italian Dry Fermented Sausage" Applied Sciences 12, no. 24: 13042. https://doi.org/10.3390/app122413042

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop