Xylosus Strains Research Articles

Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase

Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42 degrees C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC(2), pNPC(4), pNPC(10), pNPC(12), pNPC(14), pNPC(16), pNPC(18)). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95 degrees C, 77% of the initial activity was retained.

Surface migration of Staphylococcus xylosus on low-agar media

Staphylococcus xylosus is a commensal species commonly found on the skin of mammals, but also currently used as starter culture for meat fermentation. Most strains of this species colonize by forming a biofilm on abiotic surfaces. We show here that the majority of S. xylosus strains also exhibit extensive colony spreading on the surface of soft agar media. This phenomenon seemed to be independent of biofilm-forming ability. It occurred in different culture media and was dependent on temperature. Formation of a giant S. xylosus colony did not involve a biosurfactant. Microscopic observation showed that the front of the giant colony comprised a single layer of spacing cells with more packed cells in the median area. Supplementation of the soft media with DNase I increased S. xylosus colony spreading, indicating that extracellular DNA may be involved in limiting the phenomenon. The ability of S. xylosus to spread on semi-solid surfaces may constitute an advantage for surface colonization.

Characterization of Micrococcaceae isolated from Iberian dry-cured sausages

The populations of Micrococcaceae in different types of Iberian dry-cured sausages from central-west Spain were characterized and their technological and antimicrobial properties determined in order to evaluate their suitability as starter cultures in dry-cured sausage manufacture. Of a total of four hundred strains isolated from two manufacturers, one hundred and sixty-six were selected to evaluate nitrate reductase, proteolytic, lipolytic, and antimicrobial activities, and growth at different values of pH and water activity ( a w). Most of the strains were identified as Staphylococcus except for eight isolates assigned to Kocuria spp. The species most often isolated was Staphylococccus xylosus. Others were, in descending order of abundance, S. aureus, S. lugdunensis, S. saprophyticus, S. sciuri, S. chromogenes, and S. capitis. The distributions of the minority Staphylococcus species were different for the two manufacturers. All the investigated strains were able to grow at pH and a w greater than 5.0 and 0.85, respectively, the values usually found in Iberian dry-cured sausages. Five S. xylosus strains showed antimicrobial activity against some indicator strains which were investigated. Seven strains with the best properties were pre-selected and tested for their lipolytic and proteolytic activities against pork fat and myofibrillar and sarcoplasmic pork proteins, respectively, and for their low biogenic amines production. Most of the strains showed proteolytic and lipolytic activities, but none produced histamine, tyramine, phenylethylamine, or spermine. Three strains, identified as Staphylococcus xylosus, possess useful properties which make them candidates for testing as starter cultures in pilot processing of Iberian sausages.

Physical and genetic map of theStaphylococcus xylosusC2a chromosome

Staphylococcus xylosus is a ubiquitous bacterium frequently isolated from mammalian skin and occurring naturally on meat and dairy products. A physical and genetic map of the S. xylosus C2a chromosome was constructed by pulsed-field gel electrophoresis analysis after digestion with AscI, ApaI, I-CeuI, SfiI and SmaI enzymes and hybridization analysis. The chromosome size was estimated to be 2868+/-10 kb. Thirty-three genetic markers were mapped. The probable origin of replication (oriC) was positioned. Six rrn loci were located, and their orientation was determined. The chromosomes of six additional S. xylosus strains were also analysed by I-CeuI digestion, and an intraspecies diversity of the chromosome size and the number of rrn operons was shown.

Characterization of Staphylococcus xylosus and Staphylococcus carnosus isolated from Slovak meat products

The aims of this study were to isolate, identify and characterize the population of coagulase-negative staphylococci in different types of Slovak traditional sausages and to determine the metabolic properties of selected Staphylococcus xylosus and S. carnosus strains for the selection of potential starter cultures to use in the processing of sausages. The strains were tested for lactic acid production, survival in the presence of bile and sensitivity to antibiotics. Bacteriocin production, adhesion ability as well as biogenic amine (BA) production by isolates were also analysed. Most of the isolates were identified as S. xylosus and S. carnosus. Lactic acid values ranged from 0.40 to 1.03 mmol/l and strains survived in the presence of 1% bile. Most of the strains studied were sensitive to all antibiotics. Two strains, S. xylosus SO3/1M/1/2 and S. carnosus SO2/F/2/5 inhibited Listeria innocua and Pseudomonas sp. S. xylosus strains did not produce any BA, while S. carnosus SO2/F/2/5 did. S. xylosus SO3/1M/1/2 and S. carnosus SO2/F/2/5 appeared as the most adhesive strains. S. xylosus SO3/1M/1/2 with antimicrobial activity against Enterococcus avium EA5, L. innocua LMG13568 and Pseudomonas sp. SO1/1M/1/4, adhesion ability and free BA production could be used as starter culture in sausage manufacture.

Formation of biofilm by Staphylococcus xylosus

The ability of 12 Staphylococcus xylosus strains to form biofilm was determined through the study of different criteria. Eleven out of the 12 strains were able to form biofilm, 10 preferentially on hydrophilic support (glass) and one, S. xylosus C2a, on both hydrophilic and hydrophobic (polystyrene) supports. The determination of bacterial surface properties showed that all strains were negatively charged with five strains moderately hydrophobic and seven hydrophilic. The bap and icaA genes, important for biofilm formation of some staphylococci, were searched. All strains were bap positive but icaA negative. Furthermore, S. xylosus strain C2a was studied on two supports widely used in the food industry, polytetrafluoroethylene (PTFE, hydrophobic) and stainless steel (hydrophilic) and appeared to adhere preferentially on stainless steel. Addition of 20 g/l of NaCl to Tryptic Soy Broth medium (TSB) did not improve significantly its adhesion but enhanced both bacterial growth and cell survival, which were optimum in this medium. Environmental scanning electron microscopy showed that S. xylosus C2a colonized the surface of stainless steel chips with intercellular spaces. The strain formed cell aggregates embedded in an amorphous polysaccharidic matrix. Indeed, synthesis of polysaccharides increased during growth on stainless steel chips in TSB.

Ecology and characterization by molecular methods of Staphylococcus species isolated from fresh sausages

Staphylococcus spp. ecology in fresh sausages stored at 4 °C for a period of 10 days was investigated. Microbiological analyses to assess the quality and safety of the products studied were carried out, and strains on MSA agar were isolated. A total of 85 strains were identified, by traditional methods, as Staphylococcus spp. and a PCR-DGGE method, together with a S. xylosus-specific PCR, were used to determine the species of the strains isolated. Almost 50% of them were recognized as S. xylosus, but strains of S. pasteuri, S. warneri, S. equorum and S. succinus, were also found. Interesting population dynamics were observed, characterized by a succession of S. pasteuri and S. warneri at 0 day, with S. xylosus at 3 days and S. equorum at 6 and 10 days. Molecular characterization of S. xylosus strains and cluster analysis highlighted the presence of six main populations in the fresh sausages studied. However, the clusters were formed by strains isolated at different days of storage, implying a homogeneous distribution of the six subpopulations throughout the period in the products followed.

Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy

The aims of this study were to characterize the population of Micrococcaceae in different types of fermented sausages of Southern Italy and to determine the technological properties of Staphylococcus strains in order to evaluate the suitability of selected strains as starter cultures in the processing of dry fermented pork sausages. Ninety-six strains were studied to evaluate nitrate reductase, proteolytic, lipolytic and antioxidant activities as well as growth ability at different temperatures, pH’s and NaCl concentrations. All the strains were classified as Staphylococcus except for one isolate assigned to Kocuria spp. The species most often isolated were S. saprophyticus, S. xylosus and S. equorum, although they were not equally distributed within the different sausages. Other species isolated were, in descending order of abundance, S. succinus, S. warneri, S. lentus, S. vitulus, S. pasteuri, S. epidermidis, and S. haemolyticus. In general, the S. xylosus strains exhibited the best technological properties that would make them eligible as good starter cultures for fermented meat products. However, strains belonging to other species also showed good technological properties. Finally, all strains grew at 10, 15 and 20 °C, in the presence of 10% and 15% of NaCl and at pH 5.0 and 5.5. The results showed that it is possible to formulate a broad variety of staphylococcal starter cultures, adaptable to different technological conditions and sausage manufacture practices.

Development of specific PCR primers for a rapid and accurate identification of Staphylococcus xylosus, a species used in food fermentation

Twenty-seven Staphylococcus strains isolated from food and food environments were assigned to Staphylococcus xylosus by API-Staph system. But only seven isolates had similar patterns to this species when compared to the pulse-field gel electrophoresis patterns of 12 S. xylosus strains. To perform a rapid identification of the S. xylosus species, a random amplified polymorphic DNA product of 539-bp shared by all of the S. xylosus strains was used to design a pair of primers. These primers were species-specific for S. xylosus when tested by PCR on 21 staphylococci species. This specific PCR assay confirms the identification of the seven isolates identified by PFGE to S. xylosus. In conclusion, we developed specific PCR primers for a rapid and accurate identification of the S. xylosus species.

Design and Evaluation of Specific PCR Primers for Rapid and Reliable Identification of Staphylococcus xylosus Strains Isolated from Dry Fermented Sausages

Rapid and reliable identification of Staphylococcus xylosus was achieved by species-specific PCR assays. Two sets of primers, targeting on xylulokinase (xylB) and 60 kDa heat-shock protein (hsp60) genes of S. xylosus, respectively, were designed. Species-specificity of both sets of primers was evaluated by using 27 reference strains of the DSM collection, representing 23 different species of the Staphylococcus genus and 3 species of the Kocuria genus. Moreover, 90 wild strains isolated from different fermented dry sausages were included in the analysis. By using primers xylB-F and xylB-R the expected PCR fragment was obtained only when DNA from S. xylosus was used. By contrast, amplification performed by using primers xylHs-F and xylHs-R produced a single PCR fragment, of the expected length, when DNA from S. xylosus, S. haemolyticus, S. intermedius and S. kloosii were used as template. Nevertheless, AluI digestion of the xylHs-F/xylHs-R PCR fragment allowed a clear differentiation of these 4 species. The rapidity (about 4 h from DNA isolation to results) and reliability of the PCR procedures established suggests that the method may be profitably applied for specific detection and identification of S. xylosus strains.

Characterization of Micrococcaceae isolated from dry fermented sausage

One hundred Micrococcaceae strains were isolated from two types of naturally fermented dry sausages at four different stages of the ripening process in order to select the most suitable strains according to their technological characteristics including antimicrobial activity against foodborne pathogens. Identification of the isolates revealed that 91% belonged to genus Staphylococcus, whereas 5% were identified as Kocuria varians, 2% as Arthrobacter agilis and 2% as Dermacoccus nishinomiyaensis. The species most often isolated was S. saprophyticus (22%), followed by S. carnosus (20%) and S. xylosus (10%). The isolated strains of S. xylosus (10 isolates), S. carnosus (20 isolates) and K. varians (five isolates) were further characterized. A large majority of the above strains was able to grow in the presence of 10% and 15% NaCl, to reduce nitrates at 18°C and to hydrolyse casein. The majority of the strains of S. xylosus produced acetoin and 30% of them hydrolysed tributyrin. All S. xylosus strains and a large majority of S. carnosus and K. varians strains exhibited an anti-listerial activity against three Listeria monocytogenes strains.The enzymatic potential of the strains was evaluated using the API ZYM system. S. xylosus strains exhibited high β -galactosidase and esterase–lipase activities, whereas S. carnosus strains indicated strong cysteine aminopeptidase and acid phosphatase activities and K. varians indicated very weak activities of the enzymes tested. A rapid characterization of the above strains according to their enzymatic pattern could be achieved in order to follow the strains selected as starter cultures during sausage fermentation. The contribution of the selected strains to a possible inhibition of L. monocytogenes in situ on fermented meats would be of considerable interest to enhance the hygienic quality of these products.

Differentiation of Staphylococcus xylosus Strains from Italian Sausages by Antibiotyping and Low Frequency Restriction Fragment Analysis of Genomic DNA

The aim of this study was to identify the strengths and weaknesses of two typing methods, antibiogram typing and low-frequency restriction fragment analysis of genomic DNA, for the differentiation of Staphylococcus xylosus strains. Twenty-eight Staphylococcus xylosus strains isolated from Italian fermented sausages (Naples-type salami) and S. xylosus DSM 20266 (Type strain) and DSM 6179 were analysed. Antibiotyping, recorded on the basis of susceptibility testing of 16 antibiotics, allowed 21 antibiogram-types to be distinguished. Pulsed-field gel electrophoresis techniques combined with DNA digestion by infrequently cutting enzymes such as Sma I produced 22 pattern-types among the 30 strains of S. xylosus analysed. The association of antibiotyping and PFGE typing clearly distributed S. xylosus strains into 30 combinations, showing that all the strains can be differentiated. Thus, the combination of these two techniques, in spite of limitations due to instability of a phenotype-based system, actually turned out to be the most reliable methodological approach for differentiating strains of S. xylosus.

Characterization of a genetic locus essential for maltose-maltotriose utilization in Staphylococcus xylosus

A genetic locus from Staphylococcus xylosus involved in maltose-maltotriose utilization has been characterized. The chromosomal region was identified by screening a genomic library of S. xylosus in Escherichia coli for sucrose hydrolase activity. Nucleotide sequence analysis yielded two open reading frames (malR and malA) encoding proteins of 37.7 and 62.5 kDa, respectively. MalR was found to be homologous to the LacI-GalR family of transcriptional regulators, and MalA showed high similarity to yeast alpha-1,4-glucosidases and bacterial alpha-1,6-glucosidases. Inactivation of malA in the genome of S. xylosus led to a maltose-maltotriose-negative phenotype. In cell extracts of the mutant, virtually no glucose release from maltose and short maltodextrins was detectable. Inactivation of malA in a sucrose-6-phosphate hydrolase-deficient S. xylosus strain resulted in the complete loss of the residual sucrose hydrolase activity. The MalA enzyme has a clear preference for maltose but is also able to release glucose from short maltosaccharides. It cannot cleave isomaltose. Therefore, malA encodes an alpha-1,4-glucosidase or maltase, which also liberates glucose from sucrose. Subcloning experiments indicated that malA does not possess its own promoter and is cotranscribed with malR. Its expression could not be stimulated when maltose was added to the growth medium. Chromosomal inactivation of malR led to reduced maltose utilization, although alpha-glucosidase activity in the malR mutant was slightly higher than in the wild type. In the mutant strain, maltose uptake was reduced and inducibility of the transport activity was partially lost. It seems that MalR participates in the regulation of the gene(s) for maltose transport and is needed for their full expression. Thus, the malRA genes constitute an essential genetic locus for maltosaccharide utilization in S. xylosus

Evaluation of the Staph-Zym system with staphylococci isolated from bovine intramammary infections

A total of 148 staphylococci isolated from bovine intramammary infections were used to evaluate the Staph-Zym system (ROSCO, Taastrup, Denmark). The overall accuracy of the system was 91.9%. The system correctly identified all strains of Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus xylosus and 95% of Staphylococcus intermedius strains. Of 33 Staphylococcus hyicus strains, 31 (93.9%) were classified correctly by the Staph-Zym system, as well as 8 (80%) of 10 Staphylococcus chromogenes strains. All 11 Staphylococcus epidermidis strains and the 1 Staphylococcus haemolyticus strain included in the study were identified, but the Staph-Zym system had difficulty distinguishing strains of Staphylococcus warneri and Staphylococcus hominis from other species in the S. epidermidis group. The Staph-Zym system correctly identified all six S. xylosus strains and two of three Staphyloccus sciuri strains. The Staph-Zym system was considered an acceptable alternative to conventional methods for identification of bovine mammary gland isolates.

Dynamic aspects of airborne bacterial flora over an experimental area in a suburb and distribution of resistant strains to antibacterial agents among airborne staphylococci.

Airborne bacterial floras were investigated at stations A to E and P on the UOEH campus located in a suburb of the city of Kitakyushu. Bacterial collections were carried out by a filtration method using a soluble gelatin foam filter at an altitude of 1 m from the ground and viable bacterial cells were enumerated on nutrient agar plates supplemented with 50 micrograms/ml cycloheximide. At station P, where airborne bacterial composition was evaluated as a mean value of 20 repeated experiments, there were 42% Gram-positive cocci, 37% Gram-positive rods, 3.5% Bacillus strains and 5.5% Gram-negative rods. Fluctuating values of cell amounts for Gram-positive cocci and rods during these experiments suggested that in the atmospheric layer near the ground, the former was a more transient group of airborne bacteria than the latter. When airborne bacterial flora over the experimental area was surveyed by observations at six stations, the dispersion profile of bacterial flora closely corresponded to vegetating states on the surface of the ground. Over vegetating areas, airborne bacterial flora appeared in about equal amounts of Gram-positive rods and cocci, while over bare ground, Gram-positive rods appeared more abundantly than Gram-positive cocci. To investigate the distribution of drug-resistant strains among airborne bacteria, sensitivity tests to six antibiotics were carried out with S. xylosus strains isolated at stations P and E. As a result, considerable amounts of single-drug-resistant strains for TC (DOX) and EM were recognized, but the distribution of multiple-drug-resistant S. xylosus was restricted to a few strains.

Expression of human choriogonadotropin-like material in coagulase-negative Staphylococcus species

We identified 101 coagulase-negative Staphylococcus strains obtained from different laboratories, the American Type Culture Collection, and our collection, isolated from 23 patients with overt cancer and 34 normal individuals through Kloos and Schleifer conventional methods and the Staph-Ident staphylococcal system (Analytab Products, Plainview, N.Y.). In 40 strains, identity was further verified by DNA-DNA hybridization techniques. Identification revealed 39 S. epidermidis, 22 S. hominis, 8 S. haemolyticus, 9 S. capitis, 5 S. warneri, 5 S. cohnii, 8 S. saprophyticus, and 5 S. xylosus strains, all resident species found in humans. All bacteria were tested for the expression of human choriogonadotropin (hCG)-like material by the indirect fluorescein and peroxidase immunocytochemical labeling techniques by using specific antisera to the whole hormone, to its alpha and beta subunits, to the hCG beta COOH-terminal peptide, and to a monoclonal antibody to the hCG beta. The results demonstrated that the isolates from cancer patients were not unique bacteria, as has been postulated by others; the expression of immunoreactive hCG-like material is a strain, not a species, characteristic; not every bacterial strain isolated from a cancer patient is able to express the material; hCG-producing bacteria do not necessarily indicate the presence of active disease; 20% of the strains that we studied revealed a clonal variation of the expression of hCG-like material or its subunits or both as well as a variable expression of a single hCG epitope, an observation similar to that described for malignant cells; and a specific antiserum to the whole hormone with a high affinity and high sensitivity for immunocytochemistry can be a reliable reagent for screening purposes.

Untersuchungen zum Pentose- und Pentitolstoffwechsel bei Staphylococcus xylosus und Staphylococcus saprophyticus

The degradation of pentoses and xylitol, respectively is a meaningful taxonomic parameter for the distinction of the coagulase-negative staphylococci Staphylococcus xylosus and S. saprophyticus . It is, therefore, of interest to learn more about the catabolism of these C 5 -sugars and sugar alcohols. The following pathway for the breakdown of pentoses and xylitol has been found in S. xylosus and S. saprophyticus : inducible uptake of unsubstituted carbohydrates; phosphoenolpyruvate phosphotransferase system (PTS) is not involved in the uptake of pentoses and xylitol. Isomerization of pentoses and dehydration of xylitol is inducible. Phosphorylation probably occurs on ketopentoses, since activity for D-ribulokinase could be detected in D-ribose grown cells, but neither activity for pentokinases nor D-ribose-5-phosphate-isomerase could be demonstrated. Therefore, the pathway is analogous to the metabolism of pentoses and pentitol in Enterobacteriaceae , exceptionally for D-ribose, and differs from the degradation of pentitols by Lactobacillaceae . Growth with D-xylose as substrate was inhibited by xylitol due to the inhibition of the enzyme D-xylose-isomerase. Experiments with mutants should prove the value of pentose/pentitol utilization as a biochemical character for the identification of S. xylosus and S. saprophyticus . It was possible to isolate pentose-negative mutants of S. xylosus which resemble S. saprophyticus in the pentose/pentitol metabolism, but we did not succeed in isolating pentose-positive mutant of S. saprophyticus . This indicates that degradation of xylose and/or arabinose is a characteristic property fo S. xylosus strains. Pentose-negative mutants of S. xylosus lack activity for the isomerization of pentoses but they are still typical S. xylosus as could be shown by DNA-DNA-hybridization studies. DNA-homology values between wild and mutant strains were about 100%, whereas homology values between these mutant strains and a pentose-negative S. saprophyticus wild type were 35–40% corresponding to values obtained between wild types of S. xylosus and S. saprophyticus .