Equorum Strains Research Articles

Fungal-Bacterial Mutualism: Species and Strain-Dependent Simultaneous Modulation of Branched-Chain Esters and Indole Derivatives in Fermented Sausages through Metabolite Cross-Feeding.

The precise impact of species and strain diversity on fungal-bacterial interactions and the overall community functioning has remained unclear. First, our study revealed how Debaryomyces hansenii influences diverse bacteria to accumulate key metabolites in a simulated fermented food system. For flavor, D. hansenii promoted the accumulation of branched-chain esters in Staphylococcus xylosus by promoting growth and facilitating the precursor branched-chain acids transformations but hindered the accumulation of Staphylococcus equorum. Furthermore, fungal-bacterial interactions displayed diversity among S. equorum strains. For bioactive compounds, species and strain diversity of lactic acid bacteria (LAB) also influences the production of indole derivatives. Then, we investigated specific metabolic exchanges under reciprocal interaction. Amino acids, rather than vitamins, were identified as the primary drivers of the bacterial growth promotion. Moreover, precursor transformations by D. hansenii played a significant role in branched-chain esters production. Finally, a synthetic community capable of producing high concentrations of branched-chain esters and indole derivatives was successfully constructed. These results provide valuable insights into understanding and designing synthetic communities for fermented sausages.

Technological and Safety Characterization of Coagulase-Negative Staphylococci Isolated from Sardinian Fermented Sausage Made by Ovine Meat.

Ripened sheep sausages are widely consumed in Italy, particularly in Sardinia. Despite their driving role in flavor and color development, coagulase-negative staphylococci in these products have been rarely investigated. A total of 70 CoNS cultures isolated from Sardinian sheep sausages were characterized by rep-PCR and M13-RAPD typing and identified by 16S rDNA sequencing. S. xylosus and S. equorum accounted for more than 70% of the total isolates, whilst S. pasteuri (8.5%), S. succinus (2.8%), and S. haemolyticus (2.8%) were less represented. The genes encoding the synthesis of putrescine, tyramine, cadaverine, and histamine were evaluated by PCR. None of the strains hosted genes for decarboxylases, except one S. pasteuri strain that was potentially a tyramine-producer. Antibiotic resistance was evaluated, along with nitrate reductase, lipolytic, and proteolytic activity, in a pool of selected cultures. Resistance to the primary antibiotics was rather widespread. S. xylosus, S. equorum, and S. pasteuri strains were all resistant to amoxicillin and kanamycin. S. equorum strains were sensitive to all tested antibiotics. S. xylosus strains were all resistant to penicillin B. Conversely, all S. pasteuri strains were resistant to both ampicillin and penicillin B, and four out of five strains exhibited tetracycline resistance. The high variability in the production of sheep sausages makes the search for adjunct cultures of crucial relevance. According to this perspective, the characterization of the autochthonous CSN population represents the first step to approach a starter selection.

Phenotypic and genotypic assessment of iron acquisition in diverse bovine-associated non-aureus staphylococcal strains

Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

Antibiotic Resistance/Susceptibility Profiles of Staphylococcus equorum Strains from Cheese, and Genome Analysis for Antibiotic Resistance Genes.

In food, bacteria carrying antibiotic resistance genes could play a prominent role in the spread of resistance. Staphylococcus equorum populations can become large in a number of fermented foods, yet the antibiotic resistance properties of this species have been little studied. In this work, the resistance/susceptibility (R/S) profile of S. equorum strains (n = 30) from cheese to 16 antibiotics was determined by broth microdilution. The minimum inhibitory concentration (MIC) for all antibiotics was low in most strains, although higher MICs compatible with acquired genes were also noted. Genome analysis of 13 strains showed the S. equorum resistome to be composed of intrinsic mechanisms, acquired mutations, and acquired genes. As such, a plasmidic cat gene providing resistance to chloramphenicol was found in one strain; this was able to provide resistance to Staphylococcus aureus after electroporation. An msr(A) polymorphic gene was identified in five strains. The Mrs(A) variants were associated with variable resistance to erythromycin. However, the genetic data did not always correlate with the phenotype. As such, all strains harbored a polymorphic fosB/fosD gene, although only one acquired copy was associated with strong resistance to fosfomycin. Similarly, a plasmid-associated blaR1-blaZI operon encoding a penicillinase system was identified in five ampicillin- and penicillin G-susceptible strains. Identified genes not associated with phenotypic resistance further included mph(C) in two strains and norA in all strains. The antibiotic R/S status and gene content of S. equorum strains intended to be employed in food systems should be carefully determined.

Transcriptomic analysis of Staphylococcus equorum KM1031 from the high-salt fermented seafood jeotgal under chloramphenicol, erythromycin and lincomycin stresses

Staphylococcus equorum strain KM1031 is resistant to chloramphenicol, erythromycin and lincomycin. To shed light on the genetic factors underlying these antibiotic resistances, we determined the global gene expression profile of S. equorum KM1031 using RNA sequencing. During chloramphenicol, erythromycin and lincomycin treatment, 8.3% (183/2,336), 16.0% (354/2,336), and 2.9% (63/2,336) of S. equorum KM1031 genes exhibited significant differences in expression, respectively. These three antibiotics upregulated genes related to efflux and downregulated genes related to transporters. Antibiotic treatment also upregulated osmoprotectant-related genes involved in salt tolerance. To identify specific genes functionally related to antibiotic resistance, we compared the genome of strain KM1031 with those of three S. equorum strains that are sensitive to these three antibiotics. We identified three genes of particular interest: an antibiotic biosynthesis monooxygenase gene (abm, AWC34_RS01805) related to chloramphenicol resistance, an antibiotic ABC transporter ATP-binding protein gene (msr, AWC34_RS11115) related to erythromycin resistance, and a lincosamide nucleotydyltransferase gene (lnuA, AWC34_RS13300) related to lincomycin resistance. These genes were upregulated in response to the corresponding antibiotic; in particular, msr was upregulated more than fourfold by erythromycin treatment. Finally, the results of RNA sequencing were validated by quantitative real-time PCR. This transcriptomic analysis provides genetic evidence regarding antibiotic stress responses of S. equorum strain KM1031.

Detection of virulence factors in coagulase-negative Staphylococcus spp. strains isolated from Emmental cheese

ABSTRACT Food prepared with products derived from animals are involved in most cases of staphylococcal poisoning; therefore, the research of Staphylococcus spp. in Emmental cheese is more applicable. The objective of this study was to identify coagulase-negative Staphylococcus spp. (CNS) in cheese using biochemical and molecular techniques to detect the presence of nine genes responsible for the production of enterotoxins. From 180 samples analyzed, 204 CNS strains were obtained and identified as being 46 (22.6%) S. saprophyticus strains, 27 (13.2%) S. hominis spp. hominis strains, 22 (10.8%) S. sciuri strains, 21 (10.3%) S. xylosus strains, 19 (9.3%) S. epidermidis strains, 19 (9.3%) S. haemolyticus strains, 17 (8.3%) S. lentus strains, 17 (8.3%) S. warneri strains, 11 (5.4%) S. equorum strains and 5 (2.5%) S. cohnni . Using the PCRm protocol, 14 (6.9%) strains with the presence of the genes on the enterotoxin E (SEE)11 (78.6%), J (SEJ) 1 (7%), C (SEC) 1 (7%) and I (SEI) 1 (7%) were detected. Based on the results, the type of package is not interfered of growth and isolated that Staphylococcus spp. in cheese. It was observed that bacteria capacity to produce coagulase cannot be understood as an indicative of enterotoxigenicity; therefore, the CNS should be considered as a target of importance in the epidemiology of staphylococcal intoxications. It can be concluded that CNS need to be included in bacterial foodborne disease research, since the genes responsible for the production of toxins were detected and none of the studied samples presented Staphylococcus spp. counting above the limits allowed by legislation.

Technological Activities and Antibiofilm Effect of Staphylococcus Equorum Isolated from Fermented Sausage

The present study aimed at evaluating the technological properties and the ability of Staphylococcus equorum to inhibit biofilm formation by Staphylococcus aureus and Escherichia coli O157:H7. Seventeen S. equorum strains were isolated from fermented sausage and examined for their ability to grow at 10 or 15 ºC in the presence of 10 or 15% NaCl at pH values of 4.5, 5.0 or 5.5. Technological properties including catalases, superoxide dismutase, lipolytic and proteolytic activities of S. equorum strains were determined. All strains grew under the studied conditions and showed variable activities. The strain S. equorum HMA4 showed good characters of all the studied metabolic properties and had ability to grow under all studied conditions. The antagonistic effect of S. equorum HMA4 against S. aureus or E. coli O157:H7 biofilms was studied under the above conditions. There was a significant increasing in the reduction percentage of biofilm formation at pH 5.0 and 5.5 comparing with those obtained at pH 4.5. The antibiofilm effect of S. equorum HMA4 against E. coli O157:H7 was higher than its effect on biofilm of S. aureus. In conclusion, beside its role in the developing flavor and enhancing the color of the fermented foods, S. equorum HMA4 could be used as a protective starter against some foodborne pathogens in fermented foods.

Genomic insights into Staphylococcus equorum KS1039 as a potential starter culture for the fermentation of high-salt foods

BackgroundOur previous comparative genomic analysis of Staphylococcus equorum KS1039 with five S. equorum strains illuminated the genomic basis of its safety and salt tolerance. However, a comprehensive picture of the cellular components and metabolic pathways involved in the degradation of macromolecules and development of sensory properties has not been obtained for S. equorum. Therefore, in this study, we examined the general metabolism of S. equorum based on information obtained from published complete genome sequences of six S. equorum strains isolated from different niches. Additionally, the utility of strain KS1039 as a starter culture for high-salt food fermentations was examined.ResultsAll six S. equorum strains contained genes involved in glycolysis, the tricarboxylic acid cycle, and amino acid metabolic pathways, as well as color development. Moreover, the strains had the potential to produce acetoin, butanediol, and branched chain fatty acids, all of which are important flavor compounds. None of the strains contained decarboxylase genes, which are required for histamine and tyramine production. Strain KS1039 contained bacteriocin and CRISPR/Cas gene clusters, and experimental results suggested that these genes were functional in vitro.ConclusionsThe comparative genomic analysis carried out herein provides important information on the usefulness of S. equorum KS1039 as a starter culture for the fermentation of high-salt foods in terms of safety, salt tolerance, bacteriocin production, and foreign plasmid restriction.

Genomic insights into the virulence and salt tolerance of Staphylococcus equorum

To shed light on the genetic background behind the virulence and salt tolerance of Staphylococcus equorum, we performed comparative genome analysis of six S. equorum strains. Data on four previously published genome sequences were obtained from the NCBI database, while those on strain KM1031 displaying resistance to multiple antibiotics and strain C2014 causing haemolysis were determined in this study. Examination of the pan-genome of five of the six S. equorum strains showed that the conserved core genome retained the genes for general physiological processes and survival of the species. In this comparative genomic analysis, the factors that distinguish the strains from each other, including acquired genomic factors in mobile elements, were identified. Additionally, the high salt tolerance of strains enabling growth at a NaCl concentration of 25% (w/v) was attributed to the genes encoding potassium voltage-gated channels. Among the six strains, KS1039 does not possess any of the functional virulence determinants expressed in the other strains.

Genetic diversity of Staphylococcus equorum isolates from Saeu-jeotgal evaluated by multilocus sequence typing.

Staphylococcus equorum, the predominant bacterial species detected in Saeu-jeotgal, a Korean high-salt fermented seafood, is a candidate starter bacterium for Saeu-jeotgal fermentation. A multilocus sequence typing (MLST) scheme was developed to evaluate the genetic diversity and background of S. equorum strains isolated from Saeu-jeotgal. A total of 135 strains, including 117 isolates from Saeu-jeotgal, and others from Myeolchi-jeotgal, sausage, cheese and horse skin, were subjected to MLST, and the internal fragments of seven housekeeping genes, aroE, dnaJ, glpF, gmk, hsp60, mutS, and pta, were compared. This MLST scheme produced 45 sequence types (STs) and the eBURST algorithm clustered the STs into nine clonal groups and seven singletons. Clonal group 1, the major group, consisted of 30 isolates from cheese, Saeu-jeotgal and sausages, which were classified into 12 STs. The predominant ST, ST26, comprised 25 isolates and presented as a singleton. Most of the isolates from Myeolchi-jeotgal and sausages clustered on two different branches of a phylogenetic tree generated with a cluster analysis using the maximum likelihood algorithm. This MLST scheme established the genetic backgrounds of S. equorum strains isolated from different types of food. Among the housekeeping genes used for MLST, gmk had the fewest allele types and fairly low sequence identities (74.0-90.0%) within the Staphylococcus species. Therefore, sequence analyses of the gmk gene and 16S rRNA gene can be used for the accurate and rapid identification of S. equorum.

Safety and technological characterization of Staphylococcus equorum isolates from jeotgal, a Korean high-salt-fermented seafood, for starter development

To select starters for jeotgal, a traditional Korean high-salt-fermented seafood, the safety and technological properties of its predominant bacteria isolates, which were identified as Staphylococcus equorum, were assessed. Among the 185 S. equorum isolates from jeotgal, 126 ampicillin-sensitive strains were subjected to assessments for antibiotic susceptibility and safety hazards. Sixty-six out of the 126 S. equorum strains exhibited phenotypic resistances to at least one antibiotic, and their prevailing resistances were to penicillin G (34.1%), erythromycin (9.5%) and trimethoprim (9.5%). Twenty-four S. equorum strains expressed resistance to at least two antibiotics. The lnuA for lincomycin (four strains) and pbp for β-lactam (three strains) were amplified by PCR. α-Hemolytic activity was not detected from the 126 strains, and 87 strains presented δ-hemolytic activity. Among the 87 strains, three strains exhibited β-hemolytic activity. Thirty-seven strains formed a biofilm. A hemolysin gene homologous to that of Staphylococcus epidermidis was amplified from an S. equorum strain with β-hemolytic activity by PCR; however, no PCR product homologous to the previously known staphylococcal enterotoxin genes was amplified. Thirty-nine S. equorum strains cleared all of the tested safety hazards and were adopted for technological property assessments. Among these strains, 16 strains exhibited protease, lipase and nitrate reductase activities, and seven strains did not produce four types of biogenic amines. Five biogenic amine non-producers exhibited three enzyme activities. Most of the strains could grow on the agar with 20% NaCl, and 13 strains maintained growth at the 25% NaCl condition. S. equorum KS1039, which is the most applicable strain that covers the safety and technological requirements for jeotgal, can grow at the 25% NaCl condition. Through this research study, we reconfirmed the necessity of characterization in the functionality and safety of S. equorum for starter development because all of the tested phenotypic characteristics were expressed in strain-specific manners.

Characterization of coagulase-negative staphylococci isolated from Spanish dry cured meat products

Technological and safety-related properties were analyzed in a coagulase-negative staphylococci (CNS) collection isolated from Spanish dry-cured meat products in order to use them as starter cultures. The highest nitrate reductase and proteolytic activity was showed by Staphylococcus carnosus and Staphylococcus equorum. Only a few strains were able to form biofilms and the presence of the ica gene was analyzed on them. In relation to antibiotic resistance, all S. carnosus and most of the S. equorum strains were sensitive to the antibiotics tested and the presence of the blaZ gene in the β-lactamic resistant strains was studied. Biogenic amines were produced by 25% of the strains analyzed being all the S. carnosus strains tyramine producers. Taking into account the studied properties, two S. equorum strains could be selected as adequate and safe potential starter cultures for the elaboration of meat products.

Technological and safety characteristics of Staphylococcaceae isolated from Spanish traditional dry-cured sausages

The aim of this study was to determine the technological properties (nitrate reductase, proteolytic and lipolytic activities; and the ability to grow at the temperature and pH values of fermenting sausage, and at high NaCl concentrations) and safety characteristics (amino acid decarboxylase and enterotoxigenic activities) of 38 strains of Staphylococcaceae (11 of Staphylococcus epidermidis, 15 of Staphylococcus equorum, 5 of Staphylococcus pasteuri and 7 of Staphylococcus saprophyticus) isolated from Androlla and Botillo, two Spanish traditional sausages, in order to evaluate their suitability as potential starter cultures in the manufacture of these sausages. Most strains were able to grow at 10 °C, in the presence of 10% and 15% NaCl and at pH values of 5.5 and 5.0, except for S. equorum strains, growth of which was reduced at these pH values. The proteolytic activity assessed by the agar plate method showed that 89.5% and 52.6% of the strains were able to hydrolyze sarcoplasmic and myofibrillar proteins, respectively. These results were not confirmed by electrophoretic assays as only 47.2% of the strains changed the SDS-PAGE profile of actin, myosin and/or sarcoplasmic protein extracts. The assessment of the lipolytic activity by titration showed that only 21.0% of the strains can hydrolyze pork fat to any extent; whereas the profiles of the freed fatty acids were different in the different strains. Most of the strains showed decarboxylase activity against histidine, lysine, ornithine and tyrosine, but the quantities of biogenic amines produced were in most cases <25 ppm and <5 ppm for putrescine and cadaverine, respectively. Only four strains (10.5%), of S. epidermidis, produced enterotoxin C.

Prevalence of antibiotic resistance in coagulase-negative staphylococci from spontaneously fermented meat products and safety assessment for new starters

To provide new meat starter strains lacking antibiotic (AB) resistances, we explored the AB susceptibility in 116 coagulase-negative Staphylococcus (CNS) isolates from traditionally fermented sausages (n=40) manufactured with meat from conventional animal breeding, and from meat products (n=76) made from meat of animals raised in natural habitats under low- or no-antibiotic pressure. Less than 50% of these CNS isolates showed phenotypic resistances to at least one antibiotic (AB) by using microdilution assay. Resistances to penicillins and tetracycline were most often observed and could be traced back to blaZ and tet(K) genes. Prevalence of AB resistances was species-dependent and mainly found in isolates of Staphylococcus warneri (78%), Staphylococcus capitis (75%) and Staphylococcus epidermidis (67%), but only sporadically detected in Staphylococcus carnosus (27%) and Staphylococcus equorum (18%). AB resistances were more often observed in S. xylosus isolates originating from natural habitats compared to traditionally fermented sausages made from conventional meat. A selection of 101 isolates belonging to S. xylosus (n=63), S. carnosus (n=21) and S. equorum (n=17) were subsequently grouped by pulsed-field gel electrophoresis (PFGE) into strain clusters. No S. carnosus and only five S. xylosus strains were lacking AB resistances and exhibited a PFGE genotype different from commercial starters. These strains, together with 17 S. equorum strains, were further studied for safety and technological characteristics. The ability to produce biogenic amines was not detected in any strain. PCR amplifications for enterotoxin encoding genes seg–sej were detected in one, and for δ-hemolysin encoding gene hld in four S. equorum strains, but phenotypic hemolytic activity was visible for three S. xylosus and 15 S. equorum strains. Catalase and nitrate reductase activity was observed in all isolates tested; particularly S. equorum showed high nitrate reduction. In conclusion, we were able to select four new meat starter strains (two S. xylosus and two S. equorum strains) out of 116 investigated CNS, fulfilling all safety criteria including the absence of AB resistances, production of biogenic amines and genes encoding virulence factors but exhibiting high nitrate reductase and catalase activity as suitable technological characteristics. Thus, S. equorum isolates, often the dominant species in spontaneously fermented meat products, provided a prospective meat starter species exhibiting high nitrate reduction and low prevalence of AB resistances.

Use of recA gene sequence analysis for the identification of Staphylococcus equorum strains predominant on dry-cured hams

Spanish dry-cured ham is an uncooked meat product highly appreciated due to its characteristics flavour. In this study, we examined the accuracy of biochemical tests and 16S rDNA sequencing in the identification of 56 staphylococcal strains isolated during industrial Spanish dry-cured ham processes. Important differences were observed comparing genotypic and phenotypic data. Staphylococcus xylosus was the prevalent species identified by biochemical methods (87.5%), however, sequencing of the 16S rDNA resulted in an unambiguous identification of Staphylococcus equorum (73.2%) and Staphylococcus vitulinus (8.9%) strains. Reliable identification of meat staphylococci, mainly among S. xylosus and S. equorum strains could be also achieved by means of recA gene sequence comparison. Two degenerate primers previously described for lactic acid bacteria were used to amplify an internal fragment of the recA gene. This fragment was amplified from twelve staphylococcal type strains representing frequent meat species. The results indicated that recA sequencing is an adequate method to discriminate among meat staphylococci. In addition, S. xylosus and S. equorum strains could be more accurately discriminated by recA sequencing than 16S rDNA or sodA sequencing. The S. equorum sequence diversity showed at the intra-species level by recA gene sequencing confirmed the high heterogeneity described among S. equorum strains.

Genetic diversity and biofilm formation of Staphylococcus equorum isolated from naturally fermented sausages and their manufacturing environment

S. equorum is often isolated from naturally fermented sausages and from the environment of processing units. The aim of this work was first to characterize the genetic diversity of this species in a single small processing unit manufacturing traditional sausages without the use of starter cultures. One hundred and eighteen S. equorum isolates were collected from meat products and surfaces of this unit. Secondly, the capacity to form biofilm of 57 isolates of S. equorum selected from pulsed-field gel electrophoresis (PFGE) profiles was assessed to determine if this property conferred an advantage for the colonization of surfaces in the processing unit. Characterization of the isolates by PFGE analysis revealed a high diversity of the strains with 52 distinct PFGE patterns detected in this limited environment. It showed also that the exchanges between meat products and environmental surfaces could be limited or that the strains could be adapted to a specific niche as only four strains out of the 52 identified colonized both niches. The majority of the S. equorum strains formed biofilm; this was determined using a validated test on polystyrene microplates. This ability was not correlated with their origin, meat products or environmental surfaces.

Rapid and Reliable Identification of Staphylococcus equorum by a Species-Specific PCR Assay Targeting the sodA Gene

Rapid and reliable identification of Staphylococcus (S.) equorum was achieved by species-specific PCR assays. A set of primers targeting the manganese-dependent superoxide dismutase (sodA) gene of S. equorum was designed. Species-specificity of the primer set was evaluated by using a total of 112 strains (including 27 reference strains of the DSM collection), representing 26 different species of the genus Staphylococcus, 3 species of the genus Kocuria, and different strains of Macrococcus caseolyticus. By using primers SdAEqF and SdAEqR the expected PCR fragment was obtained only when DNA from S. equorum strains was used as template. 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. equorum strains.

Molecular Identification and Differentiation of Staphylococcus Species and Strains of Cheese Origin

Amplified Ribosomal-DNA Restriction Analysis (ARDRA) was used to differentiate among 12 species and 4 subspecies of the genus Staphylococcus. With a universal primer pair a 2.4 kbp PCR-product was amplified, including the 16S rDNA, the 16S-23S rDNA interspacer region, and about 500 bp of the 23S rDNA. Species-specific restriction patterns were found using the restriction enzymes HindIII and XmnI separately. Cheese related staphylococci were clearly differentiated. ARDRA results were in good agreement with results of partial sequencing of the 16S rDNA. ARDRA could fully replace the biochemical identification with ID32 Staph (BioMerieux) which was less reliable when staphylococci of cheese origin were analysed. Genomic restriction digests of cheese-related S. equorum strains by SmaI and SacI gave unique strain-specific restriction patterns which can be used to identify starter staphylococci in a complex microbial environment such as the surface of Red-Smear cheeses.

MYP04 Mycoplasma buteonis and unclassified mycoplasmas associated with conjunctivitis, sinusitis, air sac lesions and fatal septicemia in free-ranging and captive wild birds

Amplified Ribosomal-DNA Restriction Analysis (ARDRA) was used to differentiate among 12 species and 4 subspecies of the genus Staphylococcus. With a universal primer pair a 2.4 kbp PCR-product was amplified, including the 16S rDNA, the 16S-23S rDNA interspacer region, and about 500 bp of the 23S rDNA. Species-specific restriction patterns were found using the restriction enzymes Hin dIII and Xmn I separately. Cheese related staphylococci were clearly differentiated. ARDRA results were in good agreement with results of partial sequencing of the 16S rDNA. ARDRA could fully replace the biochemical identification with ID32 Staph (BioMerieux) which was less reliable when staphylococci of cheese origin were analysed. Genomic restriction digests of cheese-related S. equorum strains by Sma I and Sac I gave unique strain-specific restriction patterns which can be used to identify starter staphylococci in a complex microbial environment such as the surface of Red-Smear cheeses.

Taxonomic Characterization of Coagulase-Negative Staphylococci in Ripening Flora from Traditional French Cheeses

Summary Various methods were used to identify 145 staphylococcal strains isolated from 25 traditional types of cheeses. With the biochemical micromethod rapid gallery system. ATB 32 Staph, only 33% of the strains could be identified. All the strains unidentified by API system (98 strains) were subjected to 35 conventional biochemical and physiological tests according to the Devriese's identification scheme (1985). The results were then analyzed by a numerical method using the Sokal and Michener similarity coefficient and the 98 strains were distributed into 15 clusters at the similarity level of 60%. Of the 145 strains analyzed, 36% were identified using the association of two phenotypic methods and 26% were considered as atypical S. xylosus or S. equorum strains. An identification method based on analysis of the ribotypes was applied to 67 strains from the 15 taxa. The cellular DNA was cleaved by the Hind III and Eco RI restriction enzymes. The restriction fragments were separated by electrophoresis, blotted and probed with fluorescein labeled plasmid pBA2 containing a Bacillus subtilis gene encoding 16S rRNA. Sixty six of the 67 strains ribotyped (98.5%) gave typical known species patterns. In conclusion, 99.5% of the 145 staphylococcal cheese strains were identified. All chese strains belonged to the novobiocin-resistant CNS species and near 90% to S. equorum, S. vitulus and S. xylosus which are not widespread among human associated strains.