Susceptibility of Coagulase-negative Staphylococci to Lysostaphin and Other Antibiotics

In general, coagulase-negative staphylococci were found to be relatively less susceptible to the lytic action of lysostaphin than coagulase-positive staphylococci. To achieve, arbitrarily, a lysis greater than 75%, it was necessary to use an increased concentration of enzyme or a longer incubation period than that usually required with coagulase-positive strains. For the most part, the cultures studied were sensitive to oxacillin, cloxacillin, dicloxacillin, nafcillin, ancillin, cephalothin, cephaloridine, fusidic acid, lincomycin, novobiocin, and neomycin [median minimal inhibitory concentrations (MIC) of 1.56 mug/ml or less]. Some degree of resistance (median MIC values of 12.5 mug/ml or greater) to benzylpenicillin, ampicillin, methicillin, tetracycline, chloretetracycline, erythromycin, ristocetin, and lysostaphin was found. Ten methicillin-resistant, coagulase-negative staphylococal strains were found to be cross-resistant to all nine of the penicillins tested, but much less resistant to the two cephalosporin analogues. In several instances, some of these strains seemed to be more sensitive to benzylpenicillin and to certain of the semisynthetic penicillins than to methicillin. Of the 18 antibiotics tested with the viable plate count method, the methicillin-resistant strains were found to be the most sensitive to lincomycin and novobiocin.     

Antibiotic sensitivity of the causative agents of suppurative surgical infection]

Sensitivity of 1492 strains of the causative agents of the surgical purulent infections, i. e. pathogenic Staphylococcus, Proteus, Ps. aeruginosa and E. coli to benzylpenicillin, streptomycin, levomycetin, tetracycline, erythromycin, oleandomycin, monomycin, kanamycin, novobiocin, ampicillin, oxacillin, ceporin, gentamicin and rifampicin was studied. Gentamicin was most active against all the bacterial species tested. The staphylococci were in addition sensitive in a high percentage of the cases to rifampicin, novobiocin, ceporin, monomycin and kanamycin. The isolates of E. coli were in addition sensitive to ceporin, monomycin and kanamycin. Sensitivity of the strains of Ps. aeruginosa and Proteus was low to all of the antibiotics except gentamycin. Most of the strains of the causative agents of the surgical purulent infections were multiresistant to 4 antibiotics. The number of the staphylococcal strains sensitive to benzylpenicillin, streptomycin and levomycetin increased in 1976 as compared to 1975 on the background of a limited use of these antibiotics in clinics.     

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.     

Coagulase-Negative,Novobiocin-Resistant Staphylococci on the Skin of Animals and Man,on Meat and in Milk

The occurrence of coagulase-negative, novobiocin-resistant staphylococci, i.e. Staphylococcus cohnii, Staphylococcus saprophyticus, Staphylococcus sciuri and Staphylococcus xylosus, on the skin of animals and man has been studied. On cultures from cats, cows, dogs, guinea pigs, mice, rabbits and sheep studied, such organisms were predominant among the coagulase-negative staphylococci. From the skin of the hands of 21 of 38 persons whose professions brought them into contact with animals, e.g. inséminât ors, slaughterhouse workers and veterinarians, coagulase-negative, novobiocin-resistant staphylococci were isolated. This finding contrasted with that regarding 50 persons lacking such contacts, of whom only 1 harboured such bacteria. S. saprophyticus was isolated only from those slaughterers presenting with wounds on their hands. Coagulase-negative, novobiocin-resistant staphylococci were also isolated from every second specimen collected from the surface of meat at a slaughterhouse. No difference in the culture results could be demonstrated from specimens collected before and after cutting-up of the carcasses. Of 26 strains of coagulase-negative, DNase-negative staphylococci isolated from milk with pathological CMT, all but 5 were novobiocin-resistant. Fifteen were classified as S. xylosus, 4 as S. sciuri and 1 as S. cohnii. Of another 15 DNase-positive strains, 3 were resistant to novobiocin. Finally, clinical infections with coagulase-negative, novobiocin-resistant staphylococci in man, e.g. urinary tract infections caused by S. saprophyticus, are considered in relation to possible contagious reservoirs and modes of spread.     

Effect of an interferon preparation on increasing bacterial sensitivity to antibiotics

Under the effect of human leukocytic alpha-interferon-II for injections there was observed an increase in sensitivity of bacteria to benzylpenicillin, ristomycin, novobiocin, streptomycin, kanamycin, monomycin and erythromycin. A higher antibacterial effect of the penicillin and erythromycin was recorded only with respect to initially resistant strains of staphylococci. For the antibiotic resistant strains of staphylococci the MICs of the penicillin and erythromycin decreased on the average 17.8 and 208 times (p less than 0.001) respectively. The interferon had no effect on sensitivity of bacteria to chloramphenicol.     

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.     

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.     

Resistance to antibiotics and heavy metal salts of coagulase-positive staphylococci belonging to different biological types (ecovars)

A total of 189 strains of S. aureus isolated from cows, sheep, swines, poultry, monkeys, rabbits, foxes, and humans and 23 strains of S. intermedius isolated from minks and sables were studied. The staphylococci belonged to different biological types (according to the Hajek-Marsalek's scheme) and ecovars (according to the Meyer-Witte's scheme). The strains were studied with respect to their resistance to 10 antibiotics (benzylpenicillin, streptomycin, tetracycline, levomycetin, erythromycin, oleandomycin, neomycin, kanamycin, monomycin, and novobiocin), mercuric chloride and cadmium sulfate. As a whole the frequency of resistance to the above preparations among the staphylococci of the animal origin was not high. Differences in the frequency and range of the resistance between strains belonging to different biological types (ecovars) were shown. The highest number of the resistant cultures was detected among the strains of the biological type E (ecovar canis) and atypical strains of S. intermedius isolated from the minks. The least number of the resistant cultures was detected among the strains of the biological type C1 (ecovar bovis) isolated from the cows. It was found that almost all strains of S. intermedius were resistant to cadmium sulfate. This may be used as an additional characteristic of the species.     

Microbial biocenosis of the nasal mucosa in healthy persons and in Staphylococcus carriers

The normal microflora of the nasal mucosa in man is differentiated into main, complementary and casual microflora, taking into account its isolation rate and characteristics of microbial contamination. The main microflora of adults, in contrast to children, is represented, in addition to coagulaso-negative (CN) staphylococci, by bacteria of the genus Corynebacterium. In staphylococcal carrier state a decrease in the isolation rate and the amount of bacteria belonging to the main and complementary microflora (CN staphylococci, Micrococcus, Streptococcus, Bacillus) was observed. Moreover, in carriers the weakening of the degree of association between CN staphylococci, Micrococcus, Corynebacterium, Streptococcus in symbiotic pairs was noted. In the absence of S. aureus carrier state the main and complementary microflora was characterized by the combination of the factors of antagonism and persistence, while in carrier state the complex of colonization factors was defective in the main microflora and well pronounced in coagulase-positive staphylococcal strains and enterobacteria.     

Significance of determining novobiocin sensitivity in the taxonomy of staphylococci

Novobiocin sensitivity of 96 strains belonging to various staphylococcal species was studied. It was noted that Staph. saprophyticus significantly differed from Staph. aureus and Staph. epidermidis with respect to the above antibiotic. The MIC up to 2 micrograms/ml and the growth inhibition zones of 26--35 mm in diameter were characteristic of Staph. aureau and Staph. epidermidis, while the respective figures for most of the strains of Staph. saprophyticus were 32--64 micrograms/ml and 12--17 mm. However, 28 percent of the strains of Staph. saprophyticus did not differ with respect to their movobiocin sensitivity from the other 2 species. It is concluded that the "novobiocin test" may be used for differentiation of staphylococci, within the genera. At the same time it was shown that the method of the paper sensitivity discs compares very favourably with the method of serial dilutions in agar not only because of its simplicity and convenience of manipulation with single strains, but also of the possibility of identifying the population heterogenicity with respect to novobiocin sensitivity.     

Staphylococcus epidermidis BV: Antibiotic Resistance Patterns, Physiological Characteristics, and Bacteriophage Susceptibility

Staphylococcus epidermidis BV is a group of mannitol-fermenting coagulase-negative staphylococci characterized by multiple antibiotic resistance, very similar biochemical characteristics, and phage susceptibility. Clinical isolates belonging to this group are resistant to most antibiotics tested, including oxacillin, lincomycin, and novobiocin. The only antibiotic to which all tested strains are sensitive is vancomycin. Common biochemical traits of the tested S. epidermidis BV strains include fermentation of trehalose and ribose, phospho-β-glucosidase activity, growth on synthetic medium with amino acids as carbon source, and lack of deoxyribonuclease, phosphatase, lipase, and gelatinase activity. Some of these characteristics appear more frequently in mannitol-positive control strains than in mannitol-negative strains. S. epidermidis BV strains carry lysogenic phages with a host range restricted to this group. These phages allow the differentiation of individual strains.     

Identification of Staphylococcus from French dry sausage

Fifty-one strains of staphylococci isolated from French dry sausages were mainly identified with Staphylococcus carnosus, S. xylosus, S. warneri and S. saprophyticus. The API Staphylococcus identification system proved to be reliable for S. xylosus and S. carnosus. The identification of S. warneri and S. saprophyticus was performed by DNA-DNA hybridization. These species are better identified by taking into account not only the API Staphylococcus system but also the following characters: novobiocin and lysozyme susceptibilities, production of D-lactate. hydrolysis of tri-olein.     

Microbial Progression in Sliced Vacuum Packaged Bacon at Refrigeration Temperatures

S ummary . Three hundred and forty eight strains of micro-organisms were isolated from packaged bacon which had been cured by one or other of 2 methods (A and B) and stored at 0–10° for 7 weeks. Before storage, Micrococcus sub-group 7 (47%) and coryneform bacteria (33%) were the principal contaminants of bacon cured by method A and Micrococcus subgroup 1 (20%) and Microbacterium thermosphactum (21%), in bacon cured by method B. After 7 days at 0°, coagulase negative staphylococci accounted for 10 and 33% of the microflora in bacon A and B, respectively: other organisms were micrococci, 60% (A) and 25% (B); corynebacteria, 20% (A) and 12·5% (B), yeasts ( Torulopsis candida and T. dattila ) 5% (A) and 29% (B). In the following month at 10°, Micrococcus sub-group 5 was the dominant (43–88%) contaminant of bacon B; the incidence of Streptococcus Group N ranged from 27–36% and that of unidentified lactic acid bacteria from 23–32%. Towards the end of storage, the order of dominance was Micrococcus sub-group 3 (38–58%) and atypical streptobacteria (38%) in A; Streptococcus Group N (42%) and Gram positive rods (5–20%) in B. Staphylococci tended to die out during storage of bacons cured by the 2 methods and coagulase positive staphylococci were not isolated.     

Preliminary studies on the characterization and distribution of Staphylococcus and Micrococcus species on animal skin.

A total of 221 strains of staphylococci and 98 strains of micrococci isolated from the skins of Eastern gray squirrels, Southern flying squirrels, raccoons, opossums, squirrel monkeys, swine, sheep, horses, cattle, and dogs were characterized in a preliminary attempt to resolve their natural relationships and distribution in nature. Staphylococci demonstrating the widest host range included Staphylococcus xylosus and unnamed Staphylococcus sp. 3. Unnamed Staphylococcus sp. 2 was isolated only from sheep, Staphylococcus sp. 4 only from opossums, Staphylococcus sp. 5 only from squirrel monkeys, and Staphylococcus sp. 6 only from swine. The predominant species isolated from human skin, including S. epidermidis, S. hominis, S. haemolyticus, and S. capitis, were either not isolated or only rarely isolated from animal skin. Micrococcus varians was the predominant Micrococcus species isolated from animal skin. M. luteus was only occasionally isolated. M. lylae, M. sedentarius, M. roseus, M. kristinae, and M. nishinomiyaensis, species occasionally isolated from human skin, were not isolated from animal skin.     

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.     

Evaluation of the lipolytic activity of starter cultures for meat fermentation purposes

A modified pork fat based agar method was specially developed to determine the lipolytic activity of starter strains used for meat fermentations. The lipolytic ability of strains of lactobacilli, pediococci, staphylococci and micrococci, was examined on agar plates using different substrates and in a lipid broth model system. The screening results indicate that lipase activity was confined to strains of Staphylococcus and Micrococcus . None of the lactic acid bacteria tested showed lipolytic activity. A strain of Staphylococcus xylosus , which displayed high lipolytic activity during the screening process was examined to find its optimum conditions for lipase activity regarding pH, temperature and NaCl concentration. The lipolytic activity of seven bacterial strains was determined under optimum conditions (0% salt, pH 7 and 30 °C) and sausage conditions (3·5% salt, pH 5 and 20 °C) using fat buffer model systems. High lipolytic activity was determined for all seven strains under optimal conditions, whereas no activity was detected under fermented sausage conditions.     

Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains.     

Supplementation of enrichment broths by novobiocin for detecting Shiga toxin-producing Escherichia coli from food: a controversial use

AIMS: To investigate the assumption that usage of novobiocin (20 mg l(-1)) in Shiga toxin-producing Escherichia coli (STEC) enrichment broths could achieve false-negative results. METHODS AND RESULTS: First, the minimum inhibitory concentration (MIC) of 74 E. coli O157:H7 and 55 non-O157:H7 STEC strains to novobiocin was determined. Second, to visualize the potential impact of novobiocin on the STEC growth during the enrichment step, the growth experiments were carried out in trypticase soy broth (TSB) with and without 20 mg l(-1) of novobiocin. The MIC values varied from 32 to > 64 mg l(-1) for the 74 E. coli O157:H7 strains, and from 16 to > 64 mg l(-1) for the 55 non-O157:H7 STEC strains. The E. coli O157:H7 strains were significantly (P < 0.001) more resistant to novobiocin than the non-O157:H7 STEC strains. The present study shows that the addition of novobiocin into enrichment broths inhibits the growth of some non-O157:H7 STEC strains, and slows down the growth of some STEC strains. CONCLUSIONS: Enrichment broths supplemented by novobiocin could lead to false-negative results for detecting STEC from food. SIGNIFICANCE AND IMPACT OF THE STUDY: We strongly suggest that novobiocin should not be systematically added into enrichment broths for detecting STEC from food.     

Features of glycolysis and pentose phosphate pathway in novobiocin sensitive and novobiocin resistant staphylococci

Intensity of glycolysis and the pentose phosphate cycle in staphylococci sensitive and resistant to novobiocin was studied. The resistant variants did not practically store lactate and the activity of glycolytic enzymes i.e. hexokinase and aldolase was lowered by 15-20 and 53-59 per cent, respectively. Monoiodoacetate, a glycolysis inhibitor suppressed the glucose oxidation rate by 53.3-66.9 per cent in the sensitive variants and by 16-21.8 per cent in the resistant variants. At the same time it was characteristic of the resistant variants to increase the activity of the pentose phosphate cycle enzymes; glucose-6-phosphate dehydrogenase by 25-38.1 per cent transketolase by 21.5-27.3 per cent and transaldolase by 30-57.1 per cent. No differences in the transhydrogenase reaction kinetics of both the novobiocin sensitive and the novobiocin resistant variants were observed.