The Tellurite Reactions of Coagulase Negative Staphylococci and Micrococci

S ummary . Methods for determining the tellurite reactions of coagulase negative staphylococci and micrococci have been applied to strains causing urinary tract infections in human patients. The tellurite positive strains were assigned to Micrococcus subgroup 3 and Staphylococcus subgroup VI of the Baird-Parker (1963) classification.     

Identification of Staphylococci Isolated from Clinical Material

A total of 350 staphylococci isolated from various clinical sources were examined for bound and free coagulase, fermentation of mannitol, and deoxyribonuclease. The economical coagulase-mannitol-agar method of Esber and Faulcomer was found to be suitable for the detection of free coagulase and mannitol fermentation. A significant number of coagulase- and mannitol-negative staphylococci proved to be deoxyribonuclease-positive.     

A Comparison of Methods and the Validity of Deoxyribonuclease Tests for the Characterization of Staphylococci Isolated from Animals

Strains isolated from pigeons belonging to the coagulase-positive species Staphylococcus intermedius , coagulase-negative Staph. hyicus subsp. chromogenes strains from cattle and pigs, and Staph. aureus strains from poultry, gave weakly positive reactions in DNase plate culture tests and heat-resistant DNase tests. Staph. aureus and Staph. intermedius strains from other sources and coagulase-negative and coagulase-positive Staph. hyicus subsp. hyicus strains reacted strongly in these tests. A standardized plate culture test procedure is proposed and the use of DNase tests in the identification of staphylococci isolated from animals is discussed.     

The Egg Yolk and Lipolytic Reactions of Coagulase Positive Staphylococci

The composition of egg yolk media is important for the identification of egg yolk positive strains of coagulase positive staphylococci. Media with glucose allowed the detection of more egg yolk positive strains than those without it. However, the presence of 50% CO₂ in the incubation atmosphere allowed a greater number of egg yolk positive strains to be detected regardless of whether glucose was present or not. This was due to the suppression of lipase activity by CO₂ thus allowing total lipid separation indicative of the egg yolk phenomenon. Staphylococcus aureus strains from human nares were egg yolk positive and strongly lipolytic while strains of animal origin and epidermal necrolysis were egg yolk negative and weakly lipolytic.     

Relationships Among Coagulase, Enterotoxin, and Heat-stable Deoxyribonuclease Production by Staphylococcus aureus

The relationship between heat-stable deoxyribonuclease and coagulase production was investigated in the interest of developing more rapid diagnostic and quantitative procedures for distinguishing toxigenic and pathogenic staphylococci from closely related saprophytic organisms.     

Determining the Presence of Superantigens in Coagulase Negative Staphylococci from Humans

Superantigens (SAgs) are important virulence factors in S. aureus. Recent studies identified their presence in animal coagulase-negative staphylococci (CNS). The emergence of human-associated SAg⁺ CNS would mark a prodigious shift in virulence capabilities. We examined CNS isolates from healthy human nares and diseased individuals, and determined that no known SAgs were present.     

耐甲氧西林凝固酶阴性葡萄球菌中多种抗生素耐药基因的观察

探讨耐甲氧西林凝固酶阴性葡萄球菌(methicillin-resistant coagulase negative staphylococci,MRCNS)中多种抗生素耐药基因的分布情况,采取Kirby Bauer标准纸片扩散法(K-B纸片扩散法)对MRCNS开展药敏实验,并通过PCR检测菌种携带的抗生素耐药基因。药敏实验结果显示,96株MRCNS对庆大霉素、红霉素、莫匹罗星以及四环素的耐药性各有差异,但对氨苄西林的耐药性却达到100%,未发现对万古霉素具有耐药性的菌株;PCR检测结果显示,9种耐药基因merA、ermB、ermC、msrA、tetM、tetK、aac(6’)-aph(2")、aph(3’)-III、mupA的阳性检出率各有差异,没有扩增出vanA、ermA、vanB以及ant(6’)-I基因,但都扩增出mecA基因。结果表明,MRCNS可以同时携带多种抗生素耐药基因,属于一种多重耐药菌。     

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.     

Characterization of Staphylococci Isolated from Raw Milk

To evaluate the pathogenicity of staphylococci from bovine raw milk, the general characteristics of 775 strains isolated from 798 samples of milk were studied. The coagulase test was performed by use of rabbit plasma. Chromogenesis, mannitol fermentation, and gelatin liquefaction were investigated on Chapman's Medium 110, after 48 hr of incubation. Production of β-hemolysin, which has been considered indicative of pathogenic staphylococci of animal origin, was determined by streaking different strains on sheep blood-agar plates in the presence of a strain of Lancefield group B streptococci. Plates were incubated at 37 C for 24 hr, and strong hemolysis was produced in the zone of interaction of β-hemolysin and some substance liberated by streptococcus (CAMP test). Of 404 strains found to be coagulase-positive, 95.8% exhibited a deep-orange pigment, 76.5% produced β-hemolysin, 91.8% fermented mannitol, and 75% liquefield gelatin. Of 371 strains which gave a negative coagulase test, about 16% fermented mannitol and liquefied gelatin; none of these strains produced β-hemolysin. When results are grouped according to pigmentation and coagulase production, β-hemolysin seems to be developed by pathogenic strains of Staphylococcus aureus only. If suitability of these tests for investigation of pathogenicity is compared, production of β-hemolysin appears to be the most useful one, since no “false positive” results were found. The use of the CAMP test as a simple and rapid technique to determine production of β-hemolysin by pathogenic strains of animal staphylococci during routine bacteriological work is suggested.     

Purification and Properties of Deoxyribonuclease B From Bacillus subtilis

An exonuclease, DNase B, was isolated from Bacillus subtilis Marburg strain. Molecular weight of DNase B was estimated to be 200,000 by glycerol gradient centrifugation, however, 56,000 by SDS-polyacrylamide gel electrophoresis. This result would indicate a possibility that the enzyme consisted of an identical subunit. The enzyme was specific for single-stranded DNA, required Mg²⁺ or Mn²⁺ (5 mm) for the maximal activity, but 40% of the activity was retained in the absence of divalent cations. The enzyme was active even in the presence of EDTA. The enzyme degraded single-stranded DNA exonucleolytically, producing oligonucleotides in the direction from the 5′-end to the 3′-end.     

Apparent Spontaneous Induction of Staphylococcus aureus Isolated from Clinical Sources

Apparent spontaneous induction in staphylococcal strains from two clinical specimens was described. One of the two phages associated with these strains was found useful in typing otherwise untypable strains.     

The Vitamin Requirements of Staphylococci Isolated from Human Skin

The vitamin requirements of 46 strains representing nine species of Staphylococcus isolated from human skin together with nine authentic reference strains of these species were determined using a chemically defined medium. Strains of Staphylococcus saprophyticus and Staphylococcus cohnii were isolated on selective media. All the strains investigated required nicotinic acid and thiamine for growth. Biotin was essential or stimulatory for all coagulase negative strains except one strain of Staphylococcus capitis. Oleic acid substituted for biotin in all cases except with one strain of Staphylococcus haemolyticus. No species pattern of biotin requirement or of the ability of oleic acid to substitute for biotin was apparent. Five out of six strains of Staph. cohnii required pantothenic acid.     

Genetic Studies of Sulfadiazine-resistant and Methionine-requiring Neisseria Isolated From Clinical Material

Deoxyribonucleate (DNA) preparations were extracted from Neisseria meningitidis (four isolates from spinal fluid and blood) and N. gonorrhoeae strains, all of which were resistant to sulfadiazine upon primary isolation. These DNA preparations, together with others from in vitro mutants of N. meningitidis and N. perflava, were examined in transformation tests by using as recipient a drug-susceptible strain of N. meningitidis (Ne 15 Sul-s Met⁺) which was able to grow in a methionine-free defined medium. The sulfadiazine resistance typical of each donor was introduced into the uniform constitution of this recipient. Production of p-aminobenzoic acid was not significantly altered thereby. Transformants elicited by DNA from the N. meningitidis clinical isolates were resistant to at least 200 μg of sulfadiazine/ml, and did not show a requirement for methionine (Sul-r Met⁺). DNA from six strains of N. gonorrhoeae, which were isolated during the period of therapeutic use of sulfonamides, conveyed lower degrees of resistance and, invariably, a concurrent methionine requirement (Sul-r/Met⁻). The requirement of these transformants, and that of in vitro mutants selected on sulfadiazine-agar, was satisfied by methionine, but not by vitamin B₁₂, homocysteine, cystathionine, homoserine, or cysteine. Sul-r Met⁺ and Sul-r/Met⁻ loci could coexist in the same genome, but were segregated during transformation. On the other hand, the dual Sul-r/Met⁻ properties were not separated by recombination, but were eliminated together. DNA from various Sul-r/Met⁻ clones tested against recipients having nonidentical Sul-r/Met⁻ mutant sites yielded Sul-s Met⁺ transformants. The met locus involved is genetically complex, and will be a valuable tool for studies of genetic fine structure of members of Neisseria, and of genetic homology between species.     

Plaque Formation in Chick Embryo Fibroblast Cells by Chlamydia Isolated From Avian and Mammalian Sources

Fifteen strains of chlamydial agents, 2 Chlamydia trachomatis and 13 Chlamydia psittaci, were tested for plaque formation on chick embryo fibroblast cells. C. trachomatis strains did not form plaques; 12 strains of C. psittaci did form plaques and 1 strain did not. The distribution of plaque sizes with C. psittaci strains was studied and it was found that the 12 strains could be tentatively grouped into three plaquing types: small (<0.50 mm), intermediate (0.50 to 0.90 mm), and large (>0.90 mm). Small plaques were predominantly associated with strains M56, ET, CP3, CT4, EBA, CS1, and CT2. Intermediate size plaques were predominantly associated with strains NJ1, VT1, and H81. Large plaques were predominantly associated with strains WC and OG. Plaque size appears to be related to virulence of Chlamydia for laboratory animals.