Neutral Lipids in the Study of Relationships of Members of the Family Micrococcaceae

The organisms studied were those of the family Micrococcaceae which cannot participate in genetic exchange with Micrococcus luteus and those whose biochemical and physiological characteristics appear to bridge the genera Staphylococcus and Micrococcus. The hydrocarbon compositions of M. luteus ATCC 4698 and Micrococcus sp. ATCC 398 were shown to be similar to those previously reported for many M. luteus strains, consisting of isomers of branched monoolefins in the range C25 to C31. However, Micrococcus sp. ATCC 398 differed somewhat by having almost all C29 isomers (approximately 88% of the hydrocarbon composition). Micrococcus spp. ATCC 401 and ATCC 146 and M. roseus strains ATCC 412, ATCC 416, and ATCC 516 contained the same type of hydrocarbon patterns, but the predominant hydrocarbons were within a lower distribution range (C23 to C27), similar to Micrococcus sp. ATCC 533 previously reported. The chromatographic profile and carbon range of the hydrocarbons of an atypical strain designated M. candicans ATCC 8456 differed significantly from the hydrocarbon pattern presented above. The hydrocarbons were identified as branched and normal olefins in the range C16 to C22. Studies of several different strains of staphylococci revealed that these organisms do not contain readily detectable amounts of aliphatic hydrocarbons. The members of the family Micrococcaceae have been divided into two major groups based on the presence or absence of hydrocarbons. With the exception of M. candicans ATCC 8456, this division corresponded to the separation of these organisms according to their deoxyribonucleic acid compositions.     

Deoxyribonucleic acid base composition of some micrococci and sarcinae

The strains designated in this paper asMicrococcus lysodeikticus, M. sodonensis, M. flavus, Sarcina flava, S. pelagia, S. variabilis, S. marginata, S. subflava, S. citrea, S. lutea andStaphylococcus afermentans have similar DNA base compositions. The mole % GC (guanine plus cytosine) contents in DNA of these strains ranged from 71.8 to 73.3 as calculated from the denaturation temperature (Tm). They may be, therefore, closely related. However, at variance with Kocur and Martinec (1962) they do not seem to be identical withMicrococcus luteus (Schroeter 1872) Cohn 1872, because the neotype culture of the latter species has a different content of guanine and cytosine in its DNA (GC=66.3%). Sarcina aurantiaca, Micrococcus dentrificans andM. luteus have a similar DNA base composition. However, they are not identical as they differ from each other in several physiological characters. In the strains designated asStaphylococcus roseus andSarcina erythromyxa the content of GC varies within the range 72–72.8%. These species do not differ from each other physiologically. They form a pink pigment, reduce nitrates, do not hydrolyze casein and gelatin, and do not produce urease. They seem, therefore, to be identical, which confirms the conclusion of Kocur and Martinec (1962) who designated them asMicrococcus roseus Flügge 1886. Micrococcus conglomeratus differs significantly in DNA base composition from almost all strains of the groupM. lysodeikticus—Staphylococcus afermentans, also fromMicrococcus luteus, M. roseus andM. denitrificans. It differs fromSarcina aurantiaca only physiologically.     

Characterization of micrococcaceae isolated from salt used for Spanish dry-cured ham

The microbial flora of salt used in the production of Spanish dry-cured ham was studied. The results indicated that Micrococcaceae constituted the predominant flora. Identification of the 369 isolates belonging to the family Micrococcaceae revealed that 60% belonged to genus Staphylococcus, 25% to Micrococcus, 6% to Kocuria, 5.4% to Dermacoccus and 0.5% to Stomatococcus. The species most often isolated was Staph. xylosus (28.9%), followed by M. lylae (21.4%), Staph. equorum (18.55%) and D. nishinomiyaensis (5.4%). The results indicate that the salt during salting process of dry-cured hams offers an ecosystem suitable for the survival of the staphylococci and micrococci.     

Nuclease Production and Lysostaphin Susceptibility of Staphylococcus aureus and Other Catalase-Positive Cocci

Some strains of Staphylococcus epidermidis and Micrococcus sp. produce nucleases. However, thermal stability was shown to be unique to the nucleases of S. aureus. In addition, two micromethods for susceptibility testing to lysostaphin were more precise and convenient than anaerobic glucose fermentation in distinguishing between the genera Staphylococcus and Micrococcus.     

Deoxyribonucleic acid base composition of some members of the Micrococcaceae

Auletta, Angela E. (Catholic University, Washington, D.C.), and E. R. Kennedy. Deoxyribonucleic acid base composition of some members of the Micrococcaceae. J. Bacteriol. 92:28-34. 1966.-Thirty-seven strains from the genera Micrococcus, Staphylococcus, Gaffkya, and Sarcina were examined for deoxyribonucleic acid base composition and biochemical activity. Organisms were tested for production of catalase, coagulase, deoxyribonuclease, oxidase, phosphatase, hydrogen sulfide, indole, and acetoin; nitrate reduction; gelatin, starch, and urea hydrolysis; citrate and ammonium phosphate utilization; NaCl tolerance; growth at 10 and 45 C, and growth in litmus milk. They were tested for production of acid from dextrose and mannitol under anaerobic conditions, and for aerobic production of acid from dextrose, mannitol, lactose, sucrose, raffinose, maltose, xylose, and glycerol. Organisms could be divided into two groups on the basis of guanine-cytosine (GC) content. Group I had an average GC content of 32%, and included all organisms which produced acid from dextrose. Group II had an average GC content of 62%, and included those organisms incapable of producing acid from dextrose under anaerobic conditions. Sarcina ureae had a GC content of 43%.     

A Taxonomic Study of the Micrococcaceae

Recent proposals for new species in the genera Staphylococcus and Micrococcus prompted the present numerical taxonomic survey. The API 50E and API 20E identification strips have been used together with a few conventional tests to examine the Micrococcaceae. Many of the proposed species formed good distinct clusters and one group could be the basis for a new genus.     

Use of Shake Cultures in a Semisolid Thioglycolate Medium for Differentiating Staphylococci from Micrococci

The standard diagnostic test for differentiating staphylococci from micrococci is based on the ability of the former to produce acid anaerobically in a glucose-containing growth medium. This test has been modified to provide greater convenience, easier interpretation of results, and better correlation with deoxyribonucleic acid (DNA) base composition. In the modified test, shake cultures in Brewer's fluid thioglycolate medium with 0.3% agar added are observed for growth in the anaerobic zone of the tubes. This test was applied to 125 strains of staphylococci and micrococci, and all except two strains gave results that were consistent with other criteria. Of particular interest were eight strains of Micrococcus saprophyticus and three strains of M. lactis that have a DNA composition of 30 to 37% guanine plus cytosine (GC). All 11 of these cultures produced anaerobic growth and thus would be classified as staphylococci. Strains of M. lactis that have a high GC content in their DNA grew only aerobically. Some cultures of staphylococci produced characteristic band patterns of anaerobic growth and other cultures produced only a few anaerobic colonies from an inoculum of 10(6) to 10(7) cells. These observations suggest some interesting genetic and metabolic capabilities in such cultures.     

Deoxyribonucleic acid base composition ofMicrococcus roseus

The percentage guanine + cytosine (GC) in the DNA of 13 strains ofMicrococcus roseus has been determined. Two methods were used to analyse the base composition, namely determination of T _m value and determination of the ratio E₂₆₀/E₂₈₀ at pH 3. The percentage GC in the strains ofM. roseus ranged from 66.2 to 73.8 and was in agreement with their present taxonomic position.     

Agreement Between Deoxyribonucleic Acid Base Composition and Taxometric Classification of Gram-Positive Cocci.

Silvestri, L. G. (Università Statale, Milan, Italy), and L. R. Hill. Agreement between deoxyribonucleic acid base composition and taxometric classification of gram-positive cocci. J. Bacteriol. 90:136-140. 1965.-It had been previously proposed, from taxometric analyses, that gram-positive, catalase-positive cocci be divided into two subgroups. Thirteen strains, representative of both subgroups, were examined for deoxyribonucleic acid (DNA) base composition, determined from melting temperatures. Per cent GC (guanine + cytosine/total bases) values fell into two groups: 30.8 to 36.5% GC and 69 to 75% GC. Strains with low per cent GC values belonged to the Staphylococcus aureus-S. saprophyticus-S. lactis taxometric subgroups, and those with high per cent GC values belonged to the S. roseus-S. afermentans subgroup. The hypothetical nature of any classification is emphasized, and, in the present work, the hypothesis derived from taxometric analyses of division into two subgroups is confirmed by the study of DNA base ratios. The two subgroups correspond, respectively, to the genera Staphylococcus and Micrococcus.     

Enzyme electrophoretograms in the analysis of taxon relatedness of Micrococcus cryophilus, Branhamella catarrhalis and atypical Neisserias.

Extracts were prepared from Micrococcus cryophilus, several strains of Branhamella catarrhalis and Neisseria spp. Esterases, NADP-dependent isocitrate dehydrogenase and malate dehydrogenase activities were assayed after electrophoresis of extracts of polyacrylamide gels. Except for Neisseria perflava and N. sicca which resolved activity bands for the acetate-esterase only, the remaining bacteria exhibited species-specific esterase patterns also for the propionate and butyrate substrates. The multiple esterase patterns from B. catarrhalis ATCC25238 were qualitatively and quantitatively different from those of B. catarrhalis ATCC23246. This finding and other evidence supports a taxonomic shift of the latter to a species level of that genus. The atypical neisserias N. caviae and N. ovis appeared to exhibit an intrageneric specificity in their esterase patterns with those from B. catarrhalis but not to the other Neisseria spp. tested. The malate dehydrogenase patterns from the atypical neisserias and B. catarrhalis ATCC23246 were qualitatively similar; however, the patterns of isocitrate dehydrogenase activity were variable for these species. Micrococcus cryophilus was distinct in its esterase and dehydrogenase bands, strongly suggesting its taxon unrelatedness to the genus Branhammella or the atypical neisserias. Of the enzymes assayed, esterase proved to be the most reliable for taxonomic identifications.     

The molecular organization of the lysostaphin gene and its sequences repeated in tandem

Summary The gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51669). It was shown by NH₂-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.Abbreviations aa amino acid(s)     

A phylogenetic analysis of staphylococci, Peptococcus saccharolyticus and Micrococcus mucilaginosus

The intra- and intergeneric relationships of the genus Staphylococcus, and the phylogenetic position of Peptococcus saccharolyticus and Micrococcus (Staphylococcus salivarius), were investigated by comparative oligonucleotide cataloguing of 16S rRNA. All the staphylococci investigated form a phylogenetically coherent group at the genus level that, in addition, contains the anaerobic species Peptococcus saccharolyticus. The genus Staphylococcus belongs to the broad Bacillus-Lactobacillus-Streptococcus cluster that is defined by Gram-positive bacteria with a low DNA G+C content. Micrococcus mucilaginosus is not a genuine member of the genus Micrococcus. The binary matching coefficients between the 16S rRNA of Micrococcus mucilaginosus and those representatives of the Arthrobacter/Micrococcus group and related genera indicate that Micrococcus mucilaginosus should be regarded as a member of a new genus.     

Identification of Micrococcaceae in Clinical Bacteriology

The cellular morphology, identifying physiological characteristics, and a key to the human genera of Micrococcaceae are presented with flow charts for identification of aerobic and anaerobic isolates. These flow charts can be amended as desired, depending upon the degree of accuracy desired. Micrococcaceae isolates in a 350-bed private general hospital during a 15-week period are tabulated to show relative numbers of the different genera and species, with their probable relationship to infection or contamination. Only 11 of the 220 Micrococcaceae isolates were not Staphylococcus; no Sarcina or Peptococcus were isolated. Of the Staphylococcus isolates, 61% were S. epidermidis. Almost 18% of the S. aureus isolates were coagulase-negative. Of the S. aureus isolates, 80% of the coagulase-positive isolates were infecting agents, as were 67% of the coagulase-negative S. aureus isolates, compared to only 48% of S. epidermidis isolates. Two of four Gaffkya isolates but only one of seven Micrococcus isolates were infecting agents. If coagulase production is used as the sole criterion for speciation of staphylococci, and Micrococcus is not differentiated from Staphylococcus, the term "coagulase-negative staphylococci" does not differentiate three distinct levels of pathogenicity. Coagulase-negative S. aureus is more virulent than S. epidermidis or Gaffkya, which are more virulent than Micrococcus or Sarcina.     

Bacteriocin-producing Lactobacillus sake as starter culture in dry sausages

One hundred and fifty-two strains of Lactobacillus spp and Micrococcus spp, isolated from dry sausages, were screened for inhibitory activity. Two of the strains assayed of the genus Lactobacillus showed bactericidal activity. They were able to inhibit Listeria monocytogenes, Listeria seeligeri, Listeria innocua, Lactobacillus alimentarius and Lactobacillus bavaricus. The strains of Escherichia coli, Salmonella bradford and Salmonella newlands, Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Serratia marcescens were resistant. Their antimicrobial activity was due to peptides detectable in the culture broths and inactivated by treatment with proteolytic enzymes. Using bacteriocin-producing Lactobacillus sake as starter cultures in dry sausages could be promising in the food industry.     

Reactivity to bacterial peptidoglycans in a phagocytosis system. 1. The range of immunological specificity of Staphylococcus aureus peptidoglycan

The antigenic features of S. aureus peptidoglycan (PG) were studied in the reaction of stimulation of oxygen-dependent neutrophil metabolism, mediated by the IgG opsonins of normal human serum. The study was carried out at different taxonomic levels: the species (S. aureus), the genus (Staphylococcus), the family (Micrococcaceae), as well as in relation to remote taxons (organisms belonging to the families Streptococcaceae, Enterobacteriaceae, Neisseriaceae, to the genus Corynebacterium). All S. aureus strains were identical with respect to the specificity of their PG, essentially differing from other bacteria in this regard. After the removal of antibodies to different PG the effectiveness of PG opsonization decreased by 10.4-44.7%. Such decrease was most pronounced in experiments with the PG of streptococci (S. pyogenes, S. faecalis, S. salivarius) and Micrococcus luteus.     

A separation of staphylococci and micrococci based on serological reactivity with antiserum specific for polyglycerophosphate.

A serological reaction with the antiserum against heterophile polyglycerophosphate (PGP) was evaluated for genus level differentiation among strains of Staphylococcus and Micrococcus-Sarcina spp.. Hot saline extracts from whole cells of Staphylococcus spp. strongly reacted with the PGP antiserum, whereas those of Micrococcus-Sarcina spp. did not. Likewise, phenol-water extracts from whole cells of Micrococcus-Sarcina spp. were not reactive with the PGP antiserum, although the extracts of staphylococcal cells again gave a strong reaction with the antiserum. This study indicates that extracts from Micrococcus-Sarcina spp. have no antigen reactive with the PGP antiserum and can thus be differentiated from extracts of Staphylococcus spp. which react strongly with the PGP antiserum.     

<Emphasis Type="Italic">Staphylococcus simulans</Emphasis> recombinant lysostaphin: Production,purification, and determination of antistaphylococcal activity

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.     

Characterization of staphylococci

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.     

Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food

AIMS: To develop a multiplex PCR that allows the identification of bacteria belonging to the Staphylococcus genus and in particular to the species Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus isolated from food manufacturing plants. METHODS AND RESULTS: Five primer pairs were used in the multiplex PCR, one specific to the Staphylococcus genus and four specific to S. xylosus, S. saprophyticus, S. epidermidis and S. aureus species. All the 31 Staphylococcus reference strains yielded a specific PCR product with the genus-specific primers. Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus gave a specific PCR fragment with the corresponding species-specific primers. No amplification with the Kocuria, Macrococcus and Micrococcus strains was observed in our conditions. This multiplex PCR was performed on 30 strains of Gram-positive cocci isolated from different workshops and fermented sausages. Among them, 28 belonged to the Staphylococcus genus and 14 were identified to S. saprophyticus, four to S. xylosus, two to S. aureus and one to S. epidermidis. CONCLUSIONS: This multiplex PCR provided reliable and repeatable PCR results. It allowed the identification of a major part of the isolates, highlighting the predominance of the S. saprophyticus species in the workshops studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This tool is a useful way to screen the strains isolated from foodstuff and food environment and to monitor these species during the food processing.     

Identification and differentiation of Staphylococcus carnosus and Staphylococcus simulans by species-specific PCR assays of sodA genes

The aim of this study was to design species-specific PCR assays for rapid and reliable identification and differentiation of Staphylococcus (S.) carnosus and S. simulans strains. Two different sets of primers, targeting the manganese-dependent superoxide dismutase (sodA) gene of S. carnosus and S. simulans, respectively, were designed. Species-specificity of both sets of primers was evaluated by using 93 strains, representing 26 different species of the genus Staphylococcus, 3 species of the genus Kocuria (K.), 1 species of the genus Micrococcus (Mic.) and 1 species of the genus Macrococcus (Mac.) as reference. By using primers simF and simR the expected PCR fragment was obtained only when purified DNA from S. simulans strains was used. Amplification performed by using primers carF and carR produced a PCR fragment of the expected length, when DNA from strains of S. carnosus and S. condimenti were used as template. Nevertheless, DraI digestion of the carF/carR PCR fragment allowed a clear differentiation of strains of these two species. Species-specific PCR assays designed during this study, overcoming many of the limitations of the traditional identification procedures, can be considered a valid strategy for detection and identification of S. carnosus and S. simulans strains. The rapidity (about 4h from DNA isolation to results), the reliability and low cost of the PCR procedures established suggests that the methods may be profitably applied for specific detection and identification of S. carnosus, S. condimenti and S. simulans strains in starter cultures and meat products.