Novobiocin-resistant Staphylococci Research Articles

Manual and expert annotation of the nearly complete genome sequence of Staphylococcus sciuri strain ATCC 29059: A reference for the oxidase-positive staphylococci that supports the atypical phenotypic features of the species group

Staphylococcus sciuri is considered to be one of the most ancestral species in the natural history of the Staphylococcus genus that consists of 48 validly described species. It belongs to the basal group of oxidase-positive and novobiocin-resistant staphylococci that diverged from macrococci approximately 250 million years ago. Contrary to other groups, the S. sciuri species group has not developed host-specific colonization strategies. Genome analysis of S. sciuri ATCC 29059 provides here the first genetic basis for atypical traits that would support the switch between the free-living style and the infective state in animals and humans. From among the most remarkable features, it was noticed in this extensive study that there were a number of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS), almost twice as many as any other staphylococci, and the co-occurrence of mevalonate and non-mevalonate pathways for isoprenoid synthesis. The sequenced strain was devoid of the main virulence factors present in Staphylococcus aureus, although it exhibited numerous heme and iron acquisition systems, as well as crt and aldH genes necessary for gold pigment synthesis. The sensing and signaling networks, exemplified by a large and typical repertoire of two-component regulatory systems and a complete panel of master regulators, such as agr, rex, mgrA, rot, sarA and sarR genes, depict the background in which S. aureus virulence genes were later acquired. An additional sigma factor, a distinct set of electron transducer elements and many gene operons similar to those found in Bacillus spp. would constitute the most visible remnant links with Bacillaceae organisms.

Characterization of an extremely halotolerant Staphylococcus arlettaeHPSSN35C isolated from Dwarka Beach, India

An extremely halotolerant bacterium designated as HPSSN35C was isolated from saline soil of Dwarka beach, India. It exhibited growth over a wide range of NaCl in medium varying from 0 to 6 M. The isolate produced peach-pink pigment above ∼1.3 M NaCl. The culture was characterized using biochemical tests, bioMerieux Staph identification kit, API ID32 Staph system, and Biolog. Due to slow growth and extreme salt tolerance no ID was obtained in Biolog. Antibiotic sensitivity to various antibiotics was tested. Phenotypic characterization showed that it belonged to the novobiocin resistant staphylococci group. Analysis of 16S rRNA gene sequence comparison of 1452 base pairs showed that isolate is closely related to Staphylococcus saprophyticus group with close relationship to Staphylococcus arlettae (99% similarity). The halotolerant S. arlettae described in literature till date have been reported to tolerate 4.5 M NaCl and produce white to yellow pigment. The present study reports for the first time extremely halotolerant S. arlettae exhibiting growth up to ∼6 M NaCl and producing peach-pink pigment above ∼1.3 M NaCl.

Occurance ofStaphylococcus nepalensisstrains in different sources including human clinical material

Five isolates of coagulase-negative staphylococci were obtained from human urine, the gastrointestinal tract of squirrel monkeys, pig skin and from the environment. All key biochemical characteristics of the tested strains corresponded with the description of Staphylococcus xylosus species. However, partial 16S rRNA gene sequences obtained from analysed strains corresponded with those of Staphylococcus nepalensis reference strains, except for two strains which differed in one residue. Ribotyping with EcoRI and HindIII restriction enzymes, whole cell protein profile analysis performed by SDS-PAGE and SmaI macrorestriction analysis were used for more precise characterization and identification of the analysed strains. Obtained results showed that EcoRI and HindIII ribotyping and whole cell protein fingerprinting are suitable and reliable methods for the differentiation of S. nepalensis strains from the other novobiocin resistant staphylococci, whereas macrorestriction analysis was found to be a good tool for strain typing. The isolation of S. nepalensis is sporadic, and according to our best knowledge this study is the first report of the occurrence of this species in human clinical material as well as in other sources.

Staphylococcus equorum and Staphylococcus succinus isolated from human clinical specimens

A polyphasic identification approach was applied to a group of 11 novobiocin-resistant staphylococci isolated from human clinical materials. Phenotypic characteristics obtained by both commercial and conventional tests assigned eight strains as Staphylococcus xylosus and three strains as ambiguous S. xylosus/Staphylococcus equorum. In contrast to biotyping, ribotyping with EcoRI and HindIII restriction endonucleases and whole-cell protein fingerprinting assigned six analysed strains as S. equorum, and five strains as Staphylococcus succinus. Confirmation of the identification was done by partial 16S rRNA gene sequencing and S. equorum isolates were verified by a PCR assay targeting the sodA gene. From the data it has been implied that ribotyping and whole-cell protein analysis can be used to differentiate between the biochemically almost indistinguishable species S. xylosus, S. equorum and S. succinus. The present study confirms what is believed to be the first occurrence of S. equorum in a relevant human clinical material in the Czech Republic and describes what is believed to be the first-ever isolation of S. succinus from human clinical material.

A comparative evaluation of phenotypic and molecular methods in the identification of members of the Staphylococcus sciuri group

Members of the Staphylococcus sciuri group ( S. sciuri, S. lentus, and S. vitulinus) are coagulase-negative, novobiocin-resistant staphylococci that could be distinguished from other staphylococci on the basis of positive oxidase activity. In the present study, a scheme based on conventional methods and utilization of various carbohydrates was evaluated for the identification of oxidase-positive staphylococci, and validated using two molecular techniques. Of the 173 oxidase-positive staphylococcal tested strains, 161 were identified as S. sciuri, 9 as S. lentus, 2 as S. vitulinus, and one as S. fleurettii by our scheme. The level of agreement with tRNA intergenic length polymorphism analysis (tDNA-PCR) was high (97.5–100% correlation). The accuracy and ease of use of this protocol suggest that it may be useful and valuable in microbiological laboratories for the identification of members of this group.

Microbial succession during ripening of Naples-type salami, a southern Italian fermented sausage

Studies were carried out on the microbiological and physico-chemical changes which occurred during the ripening of five batches of Naples-type salami, manufactured without starter cultures. Salami were sampled internally and externally, and the following microbial groups were studied: lactic acid bacteria, Micrococcaceae and yeasts. The results obtained indicated that lactobacilli constituted the predominant flora, both on the surface and in the interior of the pieces throughout the ripening period. Micrococcaceae and yeasts were also found in considerable number in both locations. Characterisation of 191 lactic isolates indicated that the salami microflora was dominated by homofermentative lactobacilli; approximately 63% of them could be identified as Lactobacillus sake; 40% showing the traits of a racemase negative variant of this species, once referred to Lactobacillus bavaricus. Yeast population mainly comprised Debaryomyces strains. All the colonies grown on mannitol salt and Kranep agar were catalase-positive cocci; novobiocin-resistant staphylococci were the only Micrococcaceae found. The API Staph identification system did not prove to be reliable: 82% of the isolates remained unidentified. To achieve improved characterisation, cluster analysis was subsequently performed on this group, corroborating the existence of a fairly homogeneous group representing an intermediate variety between Staphylococcus xylosus and Staphylococcus saprophyticus that was isolated during the whole ripening process.

Staphylococcus fleurettii sp. nov., isolated from goat's milk cheeses.

A new coagulase-negative and novobiocin-resistant species of the genus Staphylococcus, Staphylococcus fleurettii, isolated from raw-milk cheeses, is described. This species is differentiated from the other novobiocin-resistant staphylococci on the basis of ribotype and intergenic transcribed spacer patterns, DNA-DNA reassociation reactions, cell wall composition and phenotypic characteristics. S. fleurettii could be distinguished by its oxidase activity, by its ability to produce acid aerobically from D-trehalose, D-mannose, D-turanose and maltose and by its inability to produce acid from D-cellobiose. The type strain of S. fleurettii is CIP 106114T (= DSM 13212T).

Stability and safety of traditional Greek salami — a microbiological ecology study

The microbiological and physicochemical changes which occurred during the industrial fermentation and ripening of four batches of Greek dry salami manufactured without starter cultures were followed. Moderated dehydration rates, monitored by slowly decreasing relative humidity from 94 to 90% during fermentation, prevented the production of insufficiently acidified batches by maintaining microbial activity for longer when the natural inoculum was low. The terminal pH values (5.0–5.2) and water contents (27.7–30.3%) of the sausages were narrowly ranged. Fermentation was governed by an active (>10 8 cfu g −1) lactic flora, consisting of `wild' strains of Lactobacillus sake. Gram-negative bacteria and aerobic sporeformers decreased below 10 2 and 10 3 cfu g −1, respectively, while yeasts did not significantly increase during ripening and were below 10 5 cfu g −1 in the ripened product. Sausages were substantially free of sulfite-reducing clostridia and coagulase-positive staphylococci during the whole process. Listeria spp., occurred in the fresh sausage mix, but disappeared from all batches at the latest by the end of fermentation. Enterococci exceeded 10 5 cfu g −1 during the first days and remained at this level during ripening. Novobiocin-resistant staphylococci matching Staphylococcus saprophyticus (mainly) and S. xylosus dominated Micrococcaceae populations, ranged between 10 5 and 10 6 cfu g −1. This is the first report of such a large contribution from S. saprophyticus to the production of dry salami of good quality. It is concluded that to keep or improve the traditional `sensory type' of Greek salamis, suitable strains of L. sake, S. xylosus and possibly nitrate-reducing S. saprophyticus should be selected and validated as starter cultures in experimentally inoculated salamis.

Staphylococcus succinus sp. nov., isolated from Dominican amber.

Two bacterial isolates, designated AMG-D1T and AMG-D2, were recovered from 25-35-million-year-old Dominican amber. AMG-D1T and AMG-D2 biochemically most closely resemble Staphylococcus xylosus; they differ physiologically from other staphylococci. Fatty acid analysis and comparisons with extensive databases were unable to show relatedness to any specific taxon. Moreover, AMG-D1T and AMG-D2 contain tuberculostearic acid and meso-diaminopimelic acid, characteristic of the G + C-rich coryneform bacteria, as opposed to L-lysine characteristic of staphylococci. AMG-D1T and AMG-D2 have a G + C ratio of 35 mol%. Phylogenetic analysis with the 16S rRNA gene indicated that AMG-D1T and AMG-D2 were most closely related to Staphylococcus equorum, S. xylosus, Staphylococcus saprophyticus and other novobiocin-resistant staphylococci. Stringent DNA-DNA hybridization studies with AMG-D1T revealed similarities of 38% with S. equorum, 23% with S. xylosus and 6% with S. saprophyticus. The results indicate that AMG-D1T and AMG-D2 represent a novel species, which was named Staphylococcus succinus sp. nov. The type strain of the new species is AMG-D1 (ATCC 700337).

Extremely halotolerant bacteria characteristic of fully cured and dried cod

Gram-positive cocci were isolated in high numbers from salted codfish during processing. They were found to be the main bacterial type in fully cured and dried salted cod. Phenotypic characterization of 37 strains showed them to belong to the novobiocin resistant staphylococci, most likely Staphylococcus arlettae or xylosus. Based on sequencing of 16S rDNA and comparison of 700 bases it was concluded that they should be assigned to the species Staphylococcus arlettae. They were found to be extremely halotolerant, growing well at salt concentrations from 0.06 M NaCl, and even displaying clear growth at 4.5 M NaCl. Likewise, the strains grew over a wide temperature range, from 8 to 45 °C. Optimal growth conditions were found to be at 0.4–0.6 M NaCl and 30–32 °C. This is all in accordance with findings for related staphylococci that have been isolated from other heavily salted meat or fish products.

Staphylococcus saprophyticus subsp. bovis subsp. nov., isolated from bovine nostrils.

A new coagulase-negative subspecies, Staphylococcus saprophyticus subsp. bovis, is described on the basis of a study of five strains isolated from the anterior nares of cows. This subspecies is differentiated from the other novobiocin-resistant staphylococci by its phenotypic properties, cell wall composition, and levels of genetic relatedness. The type strain of the new subspecies is KV 12 (=CCM 4410).

An Ecological Niche forStaphylococcus saprophyticus

Staphylococcus saprophyticus has long been associated with urinary tract infections, particularly in young women. This organism has rarely been found, in large numbers, colonising the body surface or other tissue and its ecological niche has largely remained a mystery. During studies carried out on volunteers of both sexes and of different age groups to show the numerical distribution of staphylococci from feet, some 10 000 strains were isolated and identified using a multipoint identification scheme. Novobiocin-resistant staphylococci, particularly S. cohnii , were shown to be present in concentrations of greater than 10 5 c.f.u./cm 2 on a range of individuals of both sexes, but S. saprophyticus was only isolated in these numbers from female volunteers between the ages of 10 and 29 yr. This corresponds to the peak age for urinary tract infections of which this organism is an aetiological agent. Keywords - Urinary tract infection, S. saprophyticus , Staphylococci; Feet.

A Convenient Method for Differentiation of Coagulase-Negative Staphylococci Isolated from Bovine Mammary Glands

The utility of trehalose-mannitol broth and arabinose-cellobiose broth for identification of Staphylococcus epidermidis and novobiocin-resistant staphylococci was determined using 236 coagulase-negative staphylococci isolated from bovine mammary glands. None of the 49 S. epidermidis strains was positive in trehalose-mannitol broth; whereas, all strains of Staphylococcus hyicus, Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus warneri, and Staphylococcus simulans were positive. Of the novobiocin-resistant staphylococcal species, only Staphylococcus saprophyticus was negative in arabinose-cellobiose broth. Except for one strain of Staphylococcus sciuri and one strain of Staphylococcus kloosii, all remaining strains of novobiocin-resistant staphylococcal species were positive in arabinose-cellobiose broth. Results indicate that trehalose-mannitol broth is an acceptable method for identification of S. epidermidis isolated from bovine mammary glands. Furthermore, arabinose-cellobiose broth is a useful method of screening for novobiocin-resistant staphylococci.

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.

Mannose inhibition as a significant marker for differentiating among novobiocin-resistant staphylococci of relevance in clinical microbiology.

A specific method for the identification of Staphylococcus saprophyticus among novobiocin-resistant species isolated from man is described. The test is based on novobiocin resistance, non-fermentation of D-mannose and early inhibition with late secondary growth on glucose/mannose + novobiocin agar plates. On this medium novobiocin-resistant Staphylococcus cohnii showed a confluent, continuous and homogeneous growth after 24 h which remained unchanged at 48 h whether or not it fermented D-mannose, whereas novobiocin-resistant Staphylococcus xylosus fermented D-mannose. These results are discussed in relation to a possible causal role of PTS enzymes and phosphomannose isomerase deficiency in mannose inhibition.

Staphylococcus arlettae sp. nov., S. equorum sp. nov. and S. k1oosii sp. nov.: Three New Coagulase-Negative, Novobiocin-Resistant Species from Animals

Coagulase-negative, novobiocin-resistant staphylococci isolated from various animals were allocated to three new species. Strains isolated from the skin and nares of poultry and goats belong to Staphylococcus arlettae. S. equorum comprises strains isolated from the skin of healthy horses, whereas, S. kloosii is mainly found on the skin of various wild animals and only rarely on that of farm animals. The chemical composition of the cell walls of these new species is similar to that of S. xylosus and S. saprophyticus .

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. inseminât 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.

A Study of Experimental Staphylococcal Endocarditis in Dogs

Introduction Although an enormous amount of research has been devoted to the problem of drug-resistant microbes developing from drug-sensitive strains, very little attention has been paid to changes in the opposite direction, namely the appearance of sensitive mutants arising from initially resistant microorganisms. That such a change may occur in vivo was suggested by the later disappearance of the widely prevalent sulfonamide-resistant Group A streptococci which had appeared on the scene to frustrate the initial success of the Navy's and Air Force's sulfonamide prophylaxis program during World War II. 2 In 1950 Hamburger and Berman 3 reported the replacement of streptomycinresistant Escherichia coli which had appeared in the stools of certain tuberculous patients treated with streptomycin by streptomycinsensitive E. coli after cessation of treatment and, on occasion, even during the continuation of treatment. The disappearance of novobiocin-resistant staphylococci from the noses of patients and personnel in the Chicago Municipal Contagious