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Comparison of four methods for differentiation of Staphylococcus aureus from other Micrococcaceae in the routine laboratory.

Four methods for the identification of Staphylococcus aureus (tube coagulase test, thermostable nuclease test, indirect agglutination of fibrinogen coated erythrocytes and a commercial latex kit: SeroSTAT Staphylococcus Test) have been compared. Clinical isolates (698) and 40 reference strains of Micrococcaceae were included in the study together with control organisms. The coagulase test gave no false positive results but 39/406 clinical isolates of S. aureus were negative at 2h and one half were only weakly positive. At 18 h, all but 2 of 406 isolates gave a positive reaction. The thermostable nuclease test was very specific; no clinical isolates of S. aureus gave negative results and no "coagulase-negative" clinical isolates gave a definite positive reaction. The indirect haemagglutination method was sometimes difficult to interpret and frequently gave negative or doubtful results for S. aureus. The SeroSTAT test was easy to use and interpret and was specific; the method is suitable for routine laboratory use, particularly when a rapid result is desirable.

Bacteriological Techniques↗

beta-lactamase production in coagulase-negative micrococcaceae.

A total of 8 penicillin-susceptible and 55 penicillin-resistant coagulase-negative Micrococcaceae were investigated for their beta-lactamase production by one quantitative and three qualitative methods. Seven of the 8 penicillin-susceptible strains had no beta-lactamase production; one had a very low production. Among the 55 penicillin-resistant strains, 54 produced beta-lactamase (mean 63 units/mg bacteria). Methicillin/gentamicin-resistant strains produced less enzyme than strains susceptible to these antibiotics. The percentage extracellularity (mean 18%) was lower than in Staphylococcus aureus, whereas the induction ratio (mean 56) was higher. No significant difference between the different species could be observed, but 70% of the strains were Staphylococcus epidermidis. Among the 55 beta-lactamase producing strains, the microbiological clover-leaf method detected 51, the iodometric method 40 and the chromogenic cephalosporin method 38. None of the three qualitative methods gave any false positive reactions.

Coagulase↗

Characterization of Micrococcaceae strains isolated from the human urogenital tract by the conventional scheme and a micromethod.

Two hundred and twelve Micrococcaceae isolates were obtained from 82 men with nongonococcal urethritis, 24 women with vaginitis, and 54 girls with vulvovaginitis. Identification and biotyping of these strains were carried out by using the simplified scheme of Kloos and Schleifer (W. E. Kloos and K. H. Schleifer, J. Clin. Microbiol. 1:82-88, 1975) and the commercially available API Staph test (DMS Staph Trac). Staphylococcus epidermidis occurred in about half of these isolates. There was no statistical difference between the urethral and vaginal specimens, except for S. haemolyticus found primarily in males and for S. simulans and S. aureus found primarily in girls between the ages of 1 and 12 years. S. saprophyticus, a major cause of urinary tract infections in young women, was never isolated from the vagina, suggesting the probability of another reservoir.

Bacteriological Techniques↗

Characteristics of coagulase-negative staphylococci that help differentiate these species and other members of the family Micrococcaceae.

One hundred reference strains and 1,240 clinical isolates representing 26 species of the family Micrococcaceae were used to evaluate the potential of tests for synergistic hemolysis, adherence to glass, pyroglutamyl-beta-naphthylamide hydrolysis, and susceptibility to a set of five antimicrobial agents for differentiating these species and strains within the species. Sixty-eight percent of the clinical isolates exhibited synergistic hemolysis; 69% of the clinical staphylococci but none of the micrococci or stomatococci were adherence positive, and 92% of the strong positive adherence reactions were produced by strains of Staphylococcus epidermidis. Strains from 15 of the species were pyroglutamyl-beta-naphthylamide positive, but this test separated Staphylococcus xylosus from other novobiocin-resistant staphylococci and Staphylococcus intermedius from other coagulase-positive species. A polymyxin B disk helped differentiate S. epidermidis from most other coagulase-negative staphylococci, and a bacitracin disk (10 U) helped differentiate Staphylococcus haemolyticus from most other novobiocin-susceptible staphylococci. All strains that were susceptible to furazolidone and resistant to Taxo A disks (bacitracin, 0.04 U; BBL Microbiology Systems, Cockeysville, Md.) were staphylococci. We observed a 91% correlation between species identification obtained with the Staph-Ident system (Analytab Products, Plainview, N.Y.) and conventional methods; but the micrococci and stomatococci were incorrectly identified as staphylococci with Staph-Ident, and several isolates of S. epidermidis were misidentified as Staphylococcus hominis because they were alkaline phosphatase negative. Both these problems can be prevented by adding the simple tests we describe to those already recommended when the Staph-Ident system is used to identify isolates of gram-positive, catalase-positive cocci.

Bacitracin↗

A replica-plating method for the identification of Micrococcaceae.

A procedure of replica plating is described whereby all isolated colonies of Micrococcaceae can be identified with relative ease and rapidity. The method is as accurate as the recommended procedure, but permits a more complete and economical analysis of cutaneous flora in large-scale surveys. In this system, Baird-Parker carbohydrate medium was found somewhat superior to standard medium as was incubation at 35 degrees C instead of the customary 30 degrees C. Baird-Parker's broth medium for acetoin production yielded more positive results than did commercial medium, although the reactions were less distinct. However, an agar acetoin test medium was found as good or perhaps even better than Baird-Parker's medium. The classification schemes of Baird-Parker and Bergey's Manual were contrasted in the analysis of data.

Bacteriological Techniques↗

[Value of different diagnostic tests for Micrococcaceae and their implication in pathology].

A number of 1431 products were examined and 200 coagulase-negative micrococcaceae strains were isolated and identified. On the basis of morphological aspects, enzymatic reactions and behavioural testing to antibiotics 18 strains (9%) were listed as Micrococcus and 182 (91%) as Staphylococcus strains. Efficient among the tests used for differentiating between the two genera were : microscopic aspects, pigmentogenesis, resistance to ClNa, anaerobic glucose fermentation, glycerol fermentation in the presence of erythromycin, reduction of nitrates to nitrites, and testing to novobiocin. Other tests, due to their variability, could only be used conjointly, and, finally, a further category had no diagnostic value, such as oxidase and catalase tests or the rhamnose fermentation reaction. Both the Micrococcus and the Staphylococcus strains were isolated from pathological cases and from healthy carriers, and can therefore be listed in the group of potential pathogens.

Bacterial Infections↗

[Oligodynamic action of 17 different metals on Bacillus subtilis, Enterobacteriaceae, Legionellaceae, Micrococcaceae and Pseudomonas aeruginosa].

The oligodynamic action of the pure metals aluminium, antimony, bismuth, cadmium, cobalt, copper, gold, iron, lead, manganese, mercury, nickel, platinum, silver, tin, titanium, and zinc on Bacillus subtilis (1 strain), Enterobacteriaceae (26 strains), Legionellaceae (13 strains), Micrococcaceae (6 strains), and Pseudomonas aeruginosa (4 strains) was investigated using an agar diffusion test. B. subtilis and Legionellaceae exhibited the highest susceptibility. The apathogenic micrococci and staphylococci were more susceptible to oligodynamic action of some metals than S. aureus. The group of gramnegative rods was the most resistant. The susceptibility of the different bacteria seems to be very different. Apart from the known oligodynamic action of some heavy metals the activity of antimony, cobalt, gold, and platinum should be mentioned.

Bacillus subtilis↗

Production of inhibitors of lytic activity in the Micrococcaceae.

Eight Staphylococcus xylosus strains lacking lytic activity (LA) were found to excrete agar-diffusible factors inhibiting the LA of LA-positive strains of the same species. The same eight strains, when tested against LA-positive indicator strains from other species of Micrococcaceae, caused marked LA inhibition only of strains of S, saprophyticus and S. cohnii, both species closely related to S. xylosus. Micrococci and planococci, unlike staphylococci, do not normally show LA, yet Micrococcus lylae and M. sedentarius strains produced inhibitors effective against the LA of almost all the indicator strains, and planococci inhibited the LA of a few indicator strains. Micrococcus luteus strains caused LA inhibition of homologous indicators only, i.e. exceptional M. luteus strains exhibiting LA. Thus, a specific and a non-specific type of LA inhibition can be distinguished, and it is suggested that the apparent lack of LA might, in some instances, be explained by the simultaneous production of a specific inhibitor.

Bacteriolysis↗

Consumption of antibiotics in three clinical departments and antimicrobial susceptibility of clinically significant isolates of coagulase-negative Micrococcaceae.

A total of 67 clinically significant isolates of coagulase-negative Micrococcaceae originating from blood specimens from oncologic patients, from dialysate from patients on peritoneal dialysis, and from cerebrospinal fluid from neurosurgical patients with ventricular drainage devices were identified and classified 1) according to the scheme of Baird-Parker and 2) by means of a simplified Kloos and Schleifer nomenclature. All blood isolates were classified as Staphylococcus epidermidis biotype 1 (Baird-Parker), while approximately 20% of dialysate isolates and cerebrospinal fluid isolates fell into other staphylococcal biotypes or were classified as micrococci. Using the simplified Kloos and Schleifer nomenclature, five staphylococcal isolates (8%) (four from dialysate, one from cerebrospinal fluid) were classified as "Species other than S. epidermidis". Isolates from blood exhibited a high frequency of resistance to the antibiotics tested, and 92% were found multiple-resistant (i.e. were resistant to three or more antibiotics). Isolates from cerebrospinal fluid showed multiple-resistance in 28%, while isolates from dialysate formed an intermediate group, 42% being multiple-resistant. The frequency of multiple-resistance was found to be correlated to the total consumption of antibiotics in the three clinical departments.

Anti-Bacterial Agents↗

Characterization of Micrococcaceae isolated from clinical sources.

The characterization of 556 Micrococcaceae isolates from various clinical sources has been presented. The incidence of coagulase-negative mannitol-positive staphylococci was 3.2% and that of coagulase-positive mannitol-negative staphylococci was 1.6%. There appears to be insufficient justification for the routine reporting of staphylococcal variants from a general bacteriology laboratory, and biotyping should be reserved for special circumstances.

Bacteria↗

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%.

Carbohydrate Metabolism↗

Micrococcaceae from the uinary tract in pregnancy.

Gram-positive, catalase-positive cocci (Micrococcaceae) have been obtained from the bladder by suprapubic aspiration of urine in antenatal and postnatal patients. These bacteria have been compared with similar organisms isolated from midstream specimens of urine when the suprapubic specimen was sterile. Fourteen (70%) of 20 organisms isolated from the bladder were micrococci and six (30%) were staphylococci. In contrast 17 (85%) of 20 similar organisms contaminating midstream urine specimens were staphylococci and only three (15%) were micrococci.

Female↗

Identification of psychrotrophic Micrococcaceae spp. isolated from fresh beef stored under carbon dioxide or vacuum.

Seventy-four Gram-positive, catalase-positive coccal strains were isolated from fresh beef stored under carbon dioxide (< 500 ppm O2) or vacuum for up to 15 weeks at 0, 2 or 4 degrees C. Isolates were identified using biochemical tests listed in several published protocols and the API Staph-Ident System. No isolates were identified as Staphylococcus aureus. Twenty-nine isolates were identified as Staphylococcus saprophyticus (five distinct groups), 24 isolates were identified as Staphylococcus gallinarum and 21 isolates were identified as Micrococcus varians. The staphylococcal isolates were coagulase-negative, non-hemolytic and novobiocin resistant. They produced acid from several carbohydrates under aerobic conditions, hydrolysed gelatin but not collagen, showed lipolytic activity and grew in 15% NaCl. The Micrococcus varians isolates also were salt-tolerant, produced acid only from glucose, fructose and galactose (two strains), and were resistant to lysozyme (1600 micrograms/ml). Lactic acid was the major end product of aerobic glucose metabolism. All S. saprophyticus and M. varians isolates tested contained cell wall fatty acids with chain length > or = C20:0.

Animals↗

Seasonal variation and characterization of Micrococcaceae present in ewes' raw milk.

Samples (120) of ewes' raw milk collected over a 12-month period on arrival at the dairy and freshly drawn ewes' milk samples (30) collected at the farm were analysed for total viable counts and staphylococci. Total viable counts reached on the average 5.4 X 10(6)/ml in dairy samples and 6.0 X 10(5)/ml in farm samples, whereas mean levels of staphylococci were 5.9 X 10(4)/ml and 3.3 X 10(3)/ml, respectively. A high proportion (62%) of coagulase-positive isolates from Baird-Parker agar plates was detected in spring months. Staphylococcus aureus (42%), Staph. haemolyticus (23%) and Staph. epidermidis (10%) were the predominant species in 210 characterized isolates of staphylococci from ewes' raw milk. Seven coagulase-positive, maltose-negative staphylococci could not be identified with any of the recognized Staphylococcus species. Eighteen Micrococcus strains were identified as M. varians, whereas 111 Micrococcus strains of uncertain taxonomic status were separated into three groups, according to their physiological characters.

Animals↗