Characterization of the major autolysin (AtlC) of Staphylococcus carnosus.

BACKGROUND: Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated. RESULTS: AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC. CONCLUSIONS: In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.

Control of the bifunctional O2 -sensor kinase NreB of Staphylococcus carnosus by the nitrate sensor NreA: Switching from kinase to phosphatase state.

The NreB-NreC two-component system of Staphylococcus carnosus for O2 sensing cooperates with the accessory nitrate sensor NreA in the NreA-NreB-NreC system for coordinated sensing and regulation of nitrate respiration by O2 and nitrate. ApoNreA (NreA in the absence of nitrate) interacts with NreB and inhibits NreB autophosphorylation (and activation). NreB contains the phosphatase motif DxxxQ. The present study shows that NreB on its own was inactive for the dephosphorylation of the phosphorylated response regulator NreC (NreC-P), but co-incubation with NreB and NreA stimulated NreC-P dephosphorylation. Either the presence of NreA·NO3- instead of apoNreA or mutation of the phosphatase motif (D160 or Q164) of NreB abrogated phosphatase activity of NreB. Phosphatase activity was observed for anoxic (active) NreB as well as oxic NreB, therefore the functional state of NreB is not relevant for phosphatase activity. Thus, NreB is a bifunctional sensor kinase with an integral cryptic phosphatase activity. Activation of phosphatase activity and dephosphorylation of NreC-P requires NreA as a cofactor. Accordingly, NreA and nitrate have major and dual roles in NreA-NreB-NreC regulation by (i) inhibiting NreB phosphorylation and (ii) triggering a kinase/phosphatase switch of NreB when present as apoNreA.

Application of a multiphase microreactor chemostat for the determination of reaction kinetics of Staphylococcus carnosus.

Bioreactors at the microliter scale offer a promising approach to accelerate bioprocess development. Advantages of such microbioreactors include a reduction in the use of expensive reagents. In this study, a chemostat operation mode of a cuvette-based microbubble column bioreactor made of polystyrene (working volume of 550 µL) was demonstrated. Aeration occurs through a nozzle (Ø ≤ 100 µm) and supports submerged whole-cell cultivation of Staphylococcus carnosus. Stationary concentrations of biomass and glucose were determined in the dilution rate regime ranging from 0.12 to 0.80 1/h with a glucose feed concentration of 1 g/L. For the first time, reaction kinetics of S. carnosus were estimated from data obtained from continuous cultivation. The maximal specific growth rate (µmax = 0.824 1/h), Monod constant (KS = 34 × 10- 3gS/L), substrate-related biomass yield coefficient (YX/S = 0.315 gCDW/gS), and maintenance coefficient (mS = 0.0035 gS/(gCDW·h)) were determined. These parameters are now available for further studies in the field of synthetic biology.

Secretome analysis revealed adaptive and non-adaptive responses of the Staphylococcus carnosus femB mutant.

FemABX peptidyl transferases are involved in non-ribosomal pentaglycine interpeptide bridge biosynthesis. Here, we characterized the phenotype of a Staphylococcus carnosus femB deletion mutant, which was affected in growth and showed pleiotropic effects such as enhanced methicillin sensitivity, lysostaphin resistance, cell clustering, and decreased peptidoglycan cross-linking. However, comparative secretome analysis revealed a most striking difference in the massive secretion or release of proteins into the culture supernatant in the femB mutant than the wild type. The secreted proteins can be categorized into typical cytosolic proteins and various murein hydrolases. As the transcription of the murein hydrolase genes was up-regulated in the mutant, they most likely represent an adaption response to the life threatening mutation. Even though the transcription of the cytosolic protein genes was unaltered, their high abundance in the supernatant of the mutant is most likely due to membrane leakage triggered by the weakened murein sacculus and enhanced autolysins.

Bacterial Cell Display for Selection of Affibody Molecules.

This review describes the principles for generation of affibody molecules using bacterial display on the Gram-negative Escherichia coli and the Gram-positive Staphylococcus carnosus, respectively. Affibody molecules are small and robust alternative scaffold proteins that have been explored for therapeutic, diagnostic, and biotechnological applications. They typically exhibit high-stability, affinity, and specificity with high modularity of functional domains. Due to the small size of the scaffold, affibody molecules are rapidly excreted through renal filtration and can efficiently extravasate from blood and penetrate tissues. Preclinical and clinical studies have demonstrated that affibody molecules are promising and safe complements to antibodies for in vivo diagnostic imaging and therapy. Sorting of affibody libraries displayed on bacteria using fluorescence-activated cell sorting is an effective and straightforward methodology and has been used successfully to generate novel affibody molecules with high affinity for a diverse range of molecular targets.

Effect of the protease from Staphylococcus carnosus on the proteolysis, quality characteristics, and flavor development of Harbin dry sausage.

The effect of the addition of different levels of S. carnosus protease (0, 0.15, 0.30, 0.45 and 0.60 g/kg raw meat) on the proteolysis, quality characteristics, and flavor development of Harbin dry sausage was investigated. The results showed that the S. carnosus protease addition to Harbin dry sausage effectively promoted the degradation of meat proteins into peptides and free amino acids, thus resulting in tenderization and inhibiting fat oxidation. Moreover, the S. carnosus protease addition could promote the development of key flavor compounds such as some ketones, acids and esters. Sausage with S. carnosus protease levels of 0.45 g/kg exhibited the most attractive sensory attributes. Molecular docking showed that the S. carnosus protease can interact with myosin heavy chains. In summary, the S. carnosus protease addition can improve quality characteristics and flavor profile of Harbin dry sausage.

Effect of fermentation by Pediococcus pentosaceus and Staphylococcus carnosus on the metabolite profile of sausages.

A multi-omics approach was applied to investigate the differences and correlations between characteristic volatile flavor substances and non-volatile metabolites in sausages fermented by Pediococcus pentosaceus (P. pentosaceus) and Staphylococcus carnosus (S. carnosus) alone and in a mixture. Twenty-seven volatile metabolites were identified by headspace solid-phase microextraction/gas chromatography-mass. According to orthogonal projections to latent structures-differential analysis, 17 characteristic volatile metabolites were detected in the sausages of different treatments. Utilizing ultra-high-performance liquid chromatography coupled with a mass spectrometer to analyze metabolite profiles, 42.03% of the non-volatile metabolites were classified as lipids and lipid-like molecules, 25.00% of organic acids and derivatives, and others. Seventeen characteristic flavor substances were significantly correlated with twenty differential non-volatile metabolites, and the non-volatile metabolites changed significantly. Differences in the characteristics and combinations of microorganisms themselves have a decisive role in the development of flavor substances and non-volatile metabolites in sausages.

The MpsAB Bicarbonate Transporter Is Superior to Carbonic Anhydrase in Biofilm-Forming Bacteria with Limited CO2 Diffusion.

CO2 and bicarbonate are required for carboxylation reactions, which are essential in most bacteria. To provide the cells with sufficient CO2, there exist two dissolved inorganic carbon supply (DICS) systems: the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA). Recently, it has been shown that MpsAB is a bicarbonate transporter that is present not only in photo- and autotrophic bacteria, but also in a diverse range of nonautotrophic microorganisms. Since the two systems rarely coexist in a species but are interchangeable, we investigated what advantages the one system might have over the other. Using the genus Staphylococcus as a model, we deleted the CA gene can in Staphylococcus carnosus and mpsABC genes in Staphylococcus aureus. Deletion of the respective gene in one or the other species led to growth inhibition that could only be reversed by CO2 supplementation. While the S. carnosus Δcan mutant could be fully complemented with mpsABC, the S. aureus ΔmpsABC mutant was only partially complemented by can, suggesting that MpsAB outperforms CA. Interestingly, we provide evidence that mucus biofilm formation such as that involving polysaccharide intercellular adhesin (PIA) impedes the diffusion of CO2 into cells, making MpsAB more advantageous in biofilm-producing strains or species. Coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. In conclusion, the distribution of MpsAB or CA in bacteria does not appear to be random as expression of bicarbonate transporters provides an advantage where diffusion of CO2 is impeded. IMPORTANCE CO2 and bicarbonate are required for carboxylation reactions in central metabolism and biosynthesis of small molecules in all bacteria. This is achieved by two different systems for dissolved inorganic carbon supply (DICS): these are the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA), but both rarely coexist in a given species. Here, we compared both systems and demonstrate that the distribution of MpsAB and/or CA within the phylum Firmicutes is apparently not random. The bicarbonate transporter MpsAB has an advantage in species where CO2 diffusion is hampered-for instance, in mucus- and biofilm-forming bacteria. However, coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. Given the clinical relevance of Staphylococcus in the medical environment, such findings contribute to the understanding of bacterial metabolism and thus are crucial for exploration of potential targets for antimicrobials. The knowledge gained here as exemplified by staphylococcal species could be extended to other pathogenic bacteria.

Staphylococcus aureus Genomes Harbor Only MpsAB-Like Bicarbonate Transporter but Not Carbonic Anhydrase as Dissolved Inorganic Carbon Supply System.

In recent years, it became apparent that not only autotrophic but also most other bacteria require CO2 or bicarbonate for growth. Two systems are available for the acquisition of dissolved inorganic carbon supply (DICS): the cytoplasmic localized carbonic anhydrase (CA) and the more recently described bicarbonate transporter MpsAB (membrane potential generating system). In the pathogenic species Staphylococcus aureus, there are contradictions in the literature regarding the presence of a CA or MpsAB. Here, we address these contradictions in detail. We could demonstrate by careful BLASTp analyses with 259 finished and 4,590 unfinished S. aureus genomes that S. aureus does not contain CA and that the bicarbonate transporter MpsAB is the only DICS system in this species. This finding is further supported by two further pieces of evidence: (i) mpsAB deletion mutants in four different S. aureus strains failed to grow under atmospheric air, which should not be the case if they possess CAs, since we have previously shown that both CA and MpsAB can substitute for each other, and (ii) S. aureus is completely resistant to CA inhibitors, whereas Staphylococcus carnosus, which has been shown to have only CA, was inhibited by ethoxyzolamide (EZA). Taken together, we demonstrate beyond doubt that the species S. aureus possesses only the bicarbonate transporter MpsAB as its sole DICS system. IMPORTANCE The discrepancies in the current literature and even in NCBI database, which listed some protein sequences annotated as Staphylococcus aureus carbonic anhydrase (CA), are misleading. One of the existing problems in publicly available sequence databases is the presence of incorrectly annotated genes, especially if they originated from unfinished genomes. Here, we demonstrate that some of these unfinished genomes are of poor quality and should be interpreted with caution. In the present study, we aimed to address these discrepancies and correct the current literature about S. aureus CA, considering the medical relevance of S. aureus. If left unchecked, these misleading studies and wrongly annotated genes might lead to a continual propagation of wrong annotation and, consequently, wrong interpretations and wasted time. In addition, we also show that bicarbonate transporter MpsAB-harboring bacteria are resistant to CA inhibitor, suggesting that pathogens possessing both MpsAB and CA are not treatable with CA inhibitors.

Biochemical properties of extracellular protease from Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins.

The characteristics of the extracellular protease, produced by Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins were investigated. The protease was purified by ammonium sulfate, ion exchange, and gel filtration chromatography to obtain a 20.0 kDa extracellular protease. The protease reached maximal activity at pH 9.0 and 50 °C and was stable at pH 7.0 to 11.0 and 20 to 40 °C. Its protease activity was easily inhibited in the presence of Zn2+ , Fe2+ , and Fe3+ . The enzymatic characterization of the protease revealed a Vmax 49.50 U/ml·min, Km 8.19 mg/ml, and the half-life = 28.06 min, ΔH* d  = 114.11 kJ/mol, ΔG* d  = 89.24 kJ/mol, and ΔS* d  = 77.00 J/mol·K at 50 °C. In addition, the protease hydrolyzed meat protein into small particles and produced soluble peptides. This study provides a basis for understanding the biochemical characteristics of the S. carnosus RT6 protease and its future application for fermented meat products.

Effect of starter culture and turmeric on physico-chemical quality of carabeef pastirma.

Carabeef samples were sliced, pressed, cured and divided into 6 groups. Starter cultures (Micrococcus varians M483 (MV), Staphylococcus carnosus (SC), Lactobacillus sakei (LS), M. varians M483+ Lb. sakei and Staph. carnosus + Lb. sakei) were inoculated at the dose of 10(6)-0(7)cfu/g and stored at 10 ± 1°C for 7 days. Uninoculated samples were maintained as control. Samples were then divided into 2 treatment groups. Samples of treatment 1 (T1) were smeared with a paste of turmeric followed by application of a thick layer of the paste of garlic, cumin, black pepper and red pepper whereas, samples of treatment 2 (T2) were applied with a thick layer of spices as above without turmeric. With the gradual fall in pH there was a reduction in water-holding capacity (WHC) of samples. The WHC of samples treated with SC+LS of T1 reduced to 6.3 ± 0.03 cm(2) and those inoculated with MV+LS of T2 to 6.2 ± 0.03 cm(2). The extract release volume (ERV) increased in all samples during storage. The least ERV of 11.7 and 11.6 ml were recorded in samples inoculated with MV of T1 and T2, respectively. The tyrosine (TV) and thiobarbituric acid (TBA) number of turmeric treated samples were significantly lower than non turmeric treated samples. The samples inoculated with LS had the least TV of 30.9 mg tyrosine/100 g of meat and TBA number of 0.06 mg manoladehyde/kg of meat. Samples inoculated with MV and LS of both T1 and T2 were better in physico-chemical qualities.

Potential of Bacteria from Alternative Fermented Foods as Starter Cultures for the Production of Wheat Sourdoughs.

Microbial strains for starter culture-initiated sourdough productions are commonly isolated from a fermenting flour-water mixture. Yet, starter culture strains isolated from matrices other than sourdoughs could provide the dough with interesting metabolic properties and hence change the organoleptic properties of the concomitant breads. Furthermore, the selection of sourdough starter cultures does not need to be limited to lactic acid bacteria (LAB), as other food-grade microorganisms are sometimes found in sourdoughs. Therefore, different strains belonging to LAB, acetic acid bacteria (AAB), and coagulase-negative staphylococci (CNS) that originated from different fermented food matrices (fermenting cocoa pulp-bean mass, fermented sausage, and water kefir), were examined as to their prevalence in a wheat sourdough ecosystem during 72-h fermentations. Limosilactobacillus fermentum IMDO 222 (fermented cocoa pulp-bean mass isolate) and Latilactobacillus sakei CTC 494 (fermented sausage isolate) seemed to be promising candidates as sourdough starter culture strains, as were the AAB strains Acetobacter pasteurianus IMDO 386B and Gluconobacter oxydans IMDO A845 (both isolated from fermented cocoa pulp-bean mass), due to their competitiveness in the wheat flour-water mixtures. Wheat breads made with G. oxydans IMDO A845 sourdoughs were significantly darker than reference wheat breads.

Identification and Quantitation of Hydroxy Fatty Acids in Fermented Sausage Samples.

The quality of fermented sausage is strongly influenced by its fatty acid (FA). However, the role of a defined starter culture in modifying sausage FA composition, and especially in the production of hydroxy FAs (HFAs), has not been determined. In this study, the FA compositions of sausages fermented with Latilactobacillus sakei, with L. sakei plus Staphylococcus carnosus, and with an aseptic control were characterized by liquid chromatography-mass spectrometry (MS)/MS and gas chromatography-MS. The sausages fermented with L. sakei, and with L. sakei plus S. carnosus, showed a reduced accumulation of poly and/or diunsaturated FAs and distinct composition of HFAs compared to the aseptic control. 2-HFAs were enriched via high-speed counter-current chromatography and identified uniquely in the L. sakei plus S. carnosus fermented sausage. Through lipid analyses, this study illustrated how the choice of a defined starter culture affected the observed FA metabolism in fermented sausages, facilitating the development of starter cultures or additives that impart desirable characteristics to sausage.

Chemical characterisation and histamine-forming bacteria in salted mullet roe products.

Sixteen salted mullet roe products sold in the retail markets in Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, water content, total volatile basic nitrogen (TVBN) and aerobic plate count (APC) in all samples ranged from 5.4 to 5.8, 5.1% to 7.2%, 15.4% to 27.3%, 32.0 to 69.6mg/100g and <1.0 to 7.1logCFU/g, respectively. None of these samples contained total coliform and Escherichia coli. The average content of each of the nine biogenic amines in all samples was less than 4mg/100g, and only one mullet roe sample had the histamine content (8.18mg/100g) greater than the 5.0mg/100g allowable limit suggested by the US Food and Drug Administration. Two histamine-producing bacterial strains capable of producing 10.7ppm and 9.6ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH) were identified as Staphylococcus carnosus by 16S rDNA sequencing with PCR amplification, and they were isolated from the sample with higher histamine content (8.18mg/100g).

Morphological and Dose-Dependent Study on the Effect of Methyl, Hexyl, and Dodecyl Rosmarinate on Staphylococcus carnosus LTH1502: Use of the Weibull Model.

The mechanisms of three antimicrobial rosmarinates (methyl-RE1, hexyl-RE6, and dodecyl-RE12) were investigated against Staphylococcus carnosus LTH1502. Scanning electron microscopy was used to determine the morphology of treated cells to gain information on potential changes in the site of action of compounds. The survival data obtained from antimicrobial activity assays were fitted to a nonlinear Weibull model to assess changes in inactivation behavior. Generally, esters became more effective with increasing length of the alkyl chain, resulting in a lower concentration for inhibition and inactivation. Weibull distribution parameters showed a downward concave inactivation pattern for RE1 above a critical concentration, indicative of a delayed log phase of the antimicrobial activity, with few cells being inactivated immediately after treatment and more cells being affected at later times. In contrast, esters having longer alkyl chains (RE6 and RE12) had an upward concave inactivation behavior, with more cells being inactivated immediately after addition of compounds. Cellular morphologies suggest that the antimicrobial mode of action of esters transitions from one that acts intracellularly (RE1) to one that predominately affects bacterial membrane (RE6 and RE12) due to changes in physicochemical properties of esters. Assessment that is based on the parameters of the Weibull model could, thus, be used to evaluate antimicrobial efficiency, in addition to MIC.

Pervasiveness of Staphylococcus carnosus over Staphylococcus xylosus is affected by the level of acidification within a conventional meat starter culture set-up.

Staphylococcus carnosus and Staphylococcus xylosus are commonly used, individually or in combination, within conventional starter cultures for the purposes of colour and flavour development during meat fermentation. Yet, little is known about the relative importance of both species under different processing conditions. The present study aimed at investigating the competitiveness of S. carnosus within a meat starter culture under different acidification profiles. The experimental set-up involved a gradient of decreasing experimental control but increasing realism, ranging from liquid meat fermentation models in a meat simulation medium, over solid mince-based meat fermentation models, to fermented sausage production on pilot-scale level. In general, S. carnosus gained a fitness advantage over S. xylosus in the most acidified variants of each set-up. In contrast, increasing persistence of S. xylosus was seen at the mildest acidification profiles, especially when approximating actual meat fermentation practices. Under such conditions, S. carnosus was reduced to co-prevalence in the mince-based meat fermentation models and was fully outcompeted on pilot-scale level. The latter was even the case when no S. xylosus starter culture was added, whereby S. carnosus was overpowered by staphylococci that originated from the meat background (mostly S. xylosus strains). The results of the present study suggested that conventional starter cultures behave differently when applied in different technological set-ups or using different recipes, with possible repercussions on fermented meat product quality.

Assessment of Antibiotic Susceptibility within Lactic Acid Bacteria and Coagulase-Negative Staphylococci Isolated from Hunan Smoked Pork, a Naturally Fermented Meat Product in China.

The aim of this study was to evaluate the antibiotic susceptibility of lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS) strains isolated from naturally fermented smoked pork produced in Hunan, China. A total of 48 strains were isolated by selective medium and identified at the species level by 16S rRNA gene sequencing as follows: Staphylococcus carnosus (23), Lactobacillus plantarum (12), Lactobacillus brevis (10), Lactobacillus sakei (1), Weissella confusa (1), and Weissella cibaria (1). All strains were typed by RAPD-PCR, and their susceptibility to 15 antibiotics was determined and expressed as the minimum inhibitory concentration (MIC) using agar dilution method. High resistance to penicillin G, streptomycin, gentamycin, vancomycin, chloramphenicol, norfloxacin, ciprofloxacin, kanamycin, and neomycin was found among the isolates. All the strains were sensitive to ampicillin, while the susceptibility to tetracycline, oxytetracycline, erythromycin, lincomycin, and roxithromycin varied. The presence of relevant resistance genes was investigated by PCR and sequencing, with the following genes detected: str(A), str(B), tet(O), tet(M), ere(A), and catA. Eleven strains, including 3 S. carnosus, 6 L. plantarum, and 2 L. brevis, harbored more than 3 antibiotic resistance genes. Overall, multiple antibiotic resistance patterns were widely observed in LAB and S. carnosus strains isolated from Hunan smoked pork. Risk assessment should be carried out with regard to the safe use of LAB and CNS in food production. PRACTICAL APPLICATION: We evaluated the antibiotic resistance of lactic acid bacteria and coagulase-negative staphylococci strains isolated from Chinese naturally fermented smoked pork. Our results may provide important data on establishing breakpoint standards for LAB and CNS and evaluating the safety risk of these strains for commercial use.

Genetic diversity and population structure of food-borne Staphylococcus carnosus strains.

The species Staphylococcus carnosus is a non-pathogenic representative of the coagulase negative staphylococci. Specific strains are applied as meat starter cultures. The species consists of two subspecies, S. carnosus ssp. carnosus and S. carnosus ssp. utilis. In order to place S. carnosus strains, characterized in former studies, in a genetic background that allows a typing of candidates for starter cultures, a Multilocus Sequence Typing (MLST) scheme was developed. Internal fragments of the genes tpiA, xprT, dat, gmk, glpK, narG, cstA, encoding triosephosphate isomerase, xanthine phosphoribosyltransferase, d-amino acid aminotransferase, guanylate kinase, glycerol kinase, the α-chain of the respiratory nitrate reductase, and a carbon starvation protein where chosen. Genes were selected based on their equal distribution in the genome, taxonomic value in subspecies differentiation and metabolic function. This MLST was applied to 44 S. carnosus strains, most of them previously analyzed for their suitability as starter cultures. The number of alleles varied between zero (tpiA) and five (cstA) and allowed the definition of nine sequence types (ST). ST1 was most abundant (18 strains), followed by ST2 (8) and ST4 (6). The nine STs confirmed a close relationship of all strains despite their isolation source and year, but lacked correlation with physiological activities relevant for starter cultures. The low amount of STs in the strain set lets us suggest that recombination between strains is rare. Thus, it is hypothesized that evolutionary events seem to be due to single point mutations rather than intrachromosomal recombination, and that the species possesses a clonal population structure.

In vitro modelling of simultaneous interactions of Listeria monocytogenes, Lactobacillus sakei, and Staphylococcus carnosus.

The Jameson effect model describes suppression of microorganism growth as being dependent on non-specific competition. This model was developed for simultaneous growth in a liquid medium and microbial interactions between Listeria monocytogenes, Lactobacillus sakei, and Staphylococcus carnosus with addition of NaNO2 to mimic the manufacturing process of salami for 48 h at 21°C and then for 14 days at 17°C. L. monocytogenes in the presence of L. sakei was inhibited by 2.120 log CFU/mL in the presence of NaNO2, and 1.146 log CFU/mL without NaNO2. Inhibition of L. monocytogenes cocultured with S. carnosus was 2.248 log CFU/mL at 48 h, but after 336 h was not significantly (p>0.05) different from L. monocytogenes in mono-culture. The interspecific competition parameter (ß) <1 indicated that the prevailing competition in co-cultures was intraspecific. Differentiation between 2 bacterial species interactions can be applied for use in starter cultures with pathogenic flora.

Nitrate reductase activity of Staphylococcus carnosus affecting the color formation in cured raw ham.

The influence of the nitrate reductase activity of two Staphylococcus carnosus strains used as starter cultures on the formation of nitrate, nitrite and color pigments in cured raw ham was investigated. In this context, microbiological, chemical and multivariate image analyses were carried out on cured raw hams, which were injected with different brines containing either nitrite or nitrate, with or without the S. carnosus starter cultures. During processing and storage, the viable counts of staphylococci remained constant at 6.5logcfu/g in the hams inoculated with starter cultures, while the background microbiota of the hams processed without the starter cultures developed after 14days. Those cured hams inoculated with S. carnosus LTH 7036 (high nitrate reductase activity) showed the highest decrease in nitrate and high nitrite concentrations in the end product, but were still in the range of the legal European level. The hams cured with nitrate and without starter culture or with the other strain, S. carnosus LTH 3838 (low nitrate reductase activity) showed higher residual nitrate levels and a lower nitrite content in the end product. The multivariate image analysis identified spatial and temporal differences in the meat pigment profiles of the differently cured hams. The cured hams inoculated with S. carnosus LTH 3838 showed an uncured core due to a delay in pigment formation. Therefore, the selection of starter cultures based on their nitrate reductase activity is a key point in the formation of curing compounds and color pigments in cured raw ham manufacture.