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Julie Steelandt,1 Damien Salmon,1,2 Elodie Gilbert,1 Eyad Almouazen,3 François NR Renaud,4 Laurène Roussel,1 Marek Haftek,5 Fabrice Pirot1,2 1University Claude Bernard Lyon 1, Faculty of Pharmacy, Fundamental, Clinical and Therapeutic Aspects of Skin Barrier Function, FRIPharm, Laboratoire de Pharmacie Galénique Industrielle, 2Hospital Pharmacy, FRIPharm, Hospital Edouard Herriot, Hospices Civils de Lyon, 3Laboratoire d’Automatique et de Génie des Procédés, University Claude Bernard Lyon 1, 4University Claude Bernard Lyon 1, UMR CNRS 5510/MATEIS, 5University Claude Bernard Lyon 1, Faculty of Pharmacy, Fundamental, Clinical and Therapeutic Aspects of Skin Barrier Function, FRIPharm, Laboratoire de Dermatologie, Lyon, France Abstract: Skin and mucosal infections constitute recurrent pathologies resulting from either inappropriate antiseptic procedures or a lack of efficacy of antimicrobial products. In this field, nanomaterials offer interesting antimicrobial properties (eg, long-lasting activity; intracellular and tissular penetration) as compared to conventional products. The aim of this work was to produce, by a new solvent-free process, a stable and easily freeze-dryable chlorhexidine-loaded polymeric nanocapsule (CHX-NC) suspension, and then to assess the antimicrobial properties of nanomaterials. The relevance of the process and the physicochemical properties of the CHX-NCs were examined by the assessment of encapsulation efficiency, stability of the nanomaterial suspension after 1 month of storage, and by analysis of granulometry and surface electric charge of nanocapsules. In vitro antimicrobial activities of the CHX-NCs and chlorhexidine digluconate solution were compared by measuring the inhibition diameters of two bacterial strains (Escherichia coli and Staphylococcus aureus) and one fungal strain (Candida albicans) cultured onto appropriate media. Based on the findings of this study, we report a new solvent-free process for the production of nanomaterials exhibiting antimicrobial activity, suitable stability, and easily incorporable as a new ingredient in various pharmaceutical products. Keywords: nanomaterial, nanocapsules, antiseptic, chlorhexidine, solvent-free process

In this work, our goal was to study the capability of a single metallic oxide to neutralize a chemical agent and to exhibit an antibacterial effect. We tested two types of magnesium oxides, MgO. The first MgO sample tested, which commercial data size characteristic was -325 mesh (MgO-1) destroyed in 3 h, 89.7% of paraoxon and 93.2% of 4-nitrophenol, the first degradation product. The second MgO sample, which commercial data size was <50 nm (MgO-2) neutralized in the same time, 19.5% of paraoxon and 10.9% of 4-nitrophenol. For MgO-1 no degradation products could be detected by GC-MS. MgO-1 had a bactericidal activity on Escherichia coli (6 log in 1 h), and showed a decrease of almost 3 log on a Staphylococcus aureus population in 3 h. MgO-2 caused a decrease of 2 log of a E.coli culture but had no activity against S. aureus. Neither of these two products had an activity on Bacillus subtilis spores. Analytical investigations showed that the real sizes of MgO nanoparticles were 11 nm for MgO-1 and 25 nm for MgO-2. Moreover, their crystalline structures were different. These results highlighted the importance of the size of the nanoparticles and their microscopic arrangements to detoxify chemical products and to inhibit or kill microbial strains., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Essential oils are natural products which have many interesting applications. Extraction of essential oils from plants is performed by classical and innovative methods. Numerous encapsulation processes have been developed and reported in the literature in order to encapsulate biomolecules, active molecules, nanocrystals, oils and also essential oils for various applications such as in vitro diagnosis, therapy, cosmetic, textile, food etc. Essential oils encapsulation led to numerous new formulations with new applications. This insures the protection of the fragile oil and controlled release. The most commonly prepared carriers are polymer particles, liposomes and solid lipid nanoparticles., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Pure glycerol fatty acid esters and glycerylic cyclocarbonic fatty acid esters have an amphiphilic structure, giving these biomolecules a broad range of physico-chemical and biological properties. Physico-chemical properties depend on chain lengths, odd or even carbon numbers on the chain, and glyceryl or cyclocarbonic polar heads. The spectrum of melting-point values for these molecules is large. Surface-activity is very important and through determination of the critical aggregation concentration (CAC), some fatty-acid esters are considered as solvo-surfactant biomolecules. Coupling these self-aggregation and crystallization properties, superhydrophilic surfaces were obtained. An efficient durable water repellent coating of various metallic and polymeric surfaces was allowed. Moreover, these fatty acid esters promoting superhydrophilicity showed biological activity against Gram positive, Gram negative, and yeast-like micro-organisms. Such surfaces coated by self-assembled fatty acid esters in a stable coagel state present a novel solution to surface-contamination risks from pathogen proliferation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The purpose of this study was to evaluate the possibility of using a semi-automated repetitive DNA sequences-based polymerase chain reaction (rep-PCR) for typing Pseudomonas aeruginosa isolates. rep-PCR profiles obtained by the DiversiLab system of 84 P. aeruginosa isolates from distinct epidemiological situations were obtained. rep-PCR groupings were in good agreement with the origin of these isolates. Linked rep-PCR profiles were observed for isolates recovered from a same family of cystic fibrosis (CF) patients, for the etiological agents of clustered cases of nosocomial infections, and for some isolates recovered from a same hospital room. rep-PCR and pulsed-field gel electrophoresis SpeI restricted genomic DNA (PFGE-SpeI) profiles were compared. In a few instances, rep-PCR revealed genetic divergences among isolates of a same group of PFGE-SpeI profiles. These divergences could reflect genetic drifts among closely related isolates, as illustrated by those observed between clinical and environmental isolates of a same group of PFGE-SpeI profiles. The interpretation of such differences will require further studies, but the rep-PCR analysis of P. aeruginosa diversity appeared to be an appropriate method to investigate infra-specific genetic relatedness.

Background and Objectives: Levobupivacaine in combination with sufentanil may be used for labour or postoperative regional analgesia. The risk of bacterial growth within these contained solutions for several hours at room temperature is unknown. We investigated the in vitro antimicrobial effect of levobupivacaine and sufentanil against common micro-organisms encountered during regional anaesthesia., Methods: Standardized suspensions of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli were incubated for 1, 3, 6 and 24 h at 25 degrees C, with saline (as control), sufentanil 0.5 or 0.75 microg mL-1, levobupivacaine hydrochloride 5.6 mg mL-1 and concentrations of 1.4, 2.8 and 5 mg mL-1 of levobupivacaine hydrochloride with sufentanil 0.5 microg mL-1. Colony counts were compared after 24 h incubation at 37 degrees C., Results: No bacterial growth was observed on any bacterial strain for any solution tested throughout the experiment., Conclusions: Our results suggest that solutions of levobupivacaine combined with sufentanil may be used for 24 h at room temperature during regional anaesthesia with no risk of bacterial growth.

In the last few years, regulations for biomolecule production, and especially for extraction and purification of animal molecules such as collagen, have been reinforced to ensure the sanitary safety of the materials. To be authorized to market biomaterials based on collagen, manufacturers now have to prove that at least one step of their process is described in guidelines to inactivate prion, viruses, and bacteria. The present study focuses on the inactivation step performed during the extraction and purification of porcine type I atelocollagen. We chose to determine the reduction factor of a 1 M NaOH step on porcine parvovirus and four bacterial strains inactivation. During the extraction step, we deliberately inoculated the collagen suspension with the different microorganisms tested. Then, 1 M NaOH was added to the suspension for 1 hour at 20 degrees C. We demonstrated that this treatment totally inactivated S. aureus, P. aeruginosa, C. albicans and A. niger which are bacterial strains responsible of severe human pathology. The reduction factors reached more than 4 logs for B. cereus spores and 4 logs for the porcine parvovirus. are encouraging as those two microorganisms are known to be very resistant to inactivation.

The permanent contact between the nipple part of pacifiers and the oral microflora offers ideal conditions for the development of biofilms. This study assessed the microbial contamination on the surface of 25 used pacifier nipples provided by day-care centers. Nine were made of silicone and 16 were made of latex. The biofilm was quantified using direct staining and microscopic observations followed by scraping and microorganism counting. The presence of a biofilm was confirmed on 80% of the pacifier nipples studied. This biofilm was mature for 36% of them. Latex pacifier nipples were more contaminated than silicone ones. The two main genera isolated were Staphylococcus and Candida. Our results confirm that nipples can be seen as potential reservoirs of infections. However, pacifiers do have some advantages; in particular, the potential protection they afford against sudden infant death syndrome. Strict rules of hygiene and an efficient antibiofilm cleaning protocol should be established to answer the worries of parents concerning the safety of pacifiers.

Biofilms develop inside endoscope channels even when valid endoscope reprocessing protocols are applied. The use of an efficient biocide is not sufficient if the channels are not cleaned thoroughly prior to disinfection. This study compared new anti-biofilm combinations of detachment promoting agents with a cleaning product in current use. Tests were performed using Teflon tubing and a contamination device that reproduces conditions that are prevalent during endoscopy. Products were subjected to static+brushing or dynamic treatments, and their ability to remove a preformed biofilm was assessed. The residual biofilm after treatment was assessed and compared with untreated controls. The percentage of surface covered by biofilm was measured after staining with crystal violet. Culturable bacteria levels were determined by plating the bacteria scraped from the tubing surface and counting the colony-forming units (CFU). Further tests were performed on actual endoscopes that had been contaminated artificially. Biofilm removal was confirmed by scanning electron microscopy. This study showed that the new anti-biofilm products prevented the build-up of biofilm and removed a mature biofilm (approximately 10(8)CFU/cm(2)), whereas protocols based on detergent-disinfectants containing quaternary ammonium compounds showed low efficacy as these protocols and products fixed the biofilm on the endoscope surfaces. The new procedure and agents represent a new approach to biofilm control that may improve the efficacy of endoscope reprocessing, and reduce the risk of transmitting infections.

The new Vitek 2 GN card (bioMérieux, Marcy-l'Etoile, France) was developed for better identification of fermenting and nonfermenting bacilli. This new card allows the identification of 159 taxa. A total of 426 isolates (331 fermenting and 95 nonfermenting gram-negative bacilli) belonging to 70 taxa covered by the database were evaluated. All isolates were identified in parallel with the ID 32 GN, the API 20E, and the API 20NE methods. The system correctly identified 97.4% (n=415) of the strains. Only 2.1% (n=9) needed additional testing. One strain (0.25%) was misidentified (Klebsiella pneumoniae subsp. pneumoniae), and another one (0.25%) was not identified (Morganella morganii subsp. morganii). The new GN card gives more accurate identifications overall for gram-negative bacilli when compared to the systems described in other similar studies.

Purpose: To assess anti-adhesion and/or bactericidal properties of vancomycin in vitro and to determine when these effects are detectable to estimate its relevance to perioperative antibiotic prophylaxis and analyze the efficacy of a newly designed vancomycin insert prototype for endophthalmitis prevention., Setting: University research laboratory, Lyon, France., Methods: Staphylococcus epidermidis clinical strain N890074 containing the intercellular adhesion locus ica was used as the infectious agent. Vancomycin was used at 20 microg/mL. A sterile biocompatible, biodegradable vancomycin insert, releasing 230 microg of antibiotics over 100 minutes, was designed especially for this study. To obtain bacterial killing curves, experiments were first performed in a 103 colony-forming units (CFU/mL) bacterial suspension containing no intraocular lenses (IOL). Then IOLs were incubated in the suspension, and bacterial adherence was determined using bacterial counting with and without antibiotic., Results: Vancomycin (solution and insert) had an anti-adhesion effect after 1 hour and a relevant bactericidal effect after 6 hours of incubation., Conclusions: Vancomycin used with irrigating solutions does not remain in the anterior chamber long enough to develop bactericidal effect. Even if it initially reduces bacterial adhesion, used at a drug level dropping below the bacterial minimal inhibitory concentration, it could result in a secondary increase of the adhesion of slime-producing bacteria. A sufficiently high concentration was obtained in vitro by the new sustained-release system, thereby overcoming the theoretical drawback of a short half-life within the anterior chamber. Anti-adhesion and bactericidal action of vancomycin inserts remains to be confirmed in clinical studies.

Bacterial adhesion to intraocular lenses (IOLs) takes place during their implantation. This is a prominent etiological factor of postoperative endophthalmitis. Following adhesion, secretion of an extracellular matrix (called slime for Staphylococcus epidermidis) and formation of multiple layers of microcolonies lead to the colonization of the biomaterial surface. Scanning electron microscopy photographs illustrate the different steps of biofilm formation. The different adhesins expressed by S. epidermidis involved in the adhesion process are described. The biofilm is not only an adhesive medium; it also affects virulence. Last, notions on biofilm physiology are discussed in an attempt to explain the dynamic equilibrium of this system. In 2004, the perfect biomaterial able to prevent postoperative endophthalmitis does not yet exist. Moreover, there is no effective tool, at the present time, to fight against mature biofilms. Therefore, preventing biofilm formation remains capital, which requires perfect knowledge of all stages of formation and the factors involved.

In the present study, an artificial neural network was trained with the Stuttgart Neural Networks Simulator, in order to identify Corynebacterium species by analyzing their pyrolysis patterns. An earlier study described the combination of pyrolysis, gas chromatography and atomic emission detection we used on whole cell bacteria. Carbon, sulfur and nitrogen were detected in the pyrolysis compounds. Pyrolysis patterns were obtained from 52 Corynebacterium strains belonging to 5 close species. These data were previously analyzed by Euclidean distances calculation followed by Unweighted Pair Group Method of Averages, a clustering method. With this early method, strains from 3 of the 5 species (C. xerosis, C. freneyi and C. amycolatum) were correctly characterized even if the 29 strains of C. amycolatum were grouped into 2 subgroups. Strains from the 2 remaining species (C. minutissimum and C. striatum) cannot be separated. To build an artificial neural network, able to discriminate the 5 previous species, the pyrolysis data of 42 selected strains were used as learning set and the 10 remaining strains as testing set. The chosen learning algorithm was Back-Propagation with Momentum. Parameters used to train a correct network are described here, and the results analyzed. The obtained artificial neural network has the following cone-shaped structure: 144 nodes in input, 25 and 9 nodes in 2 successive hidden layers, and then 5 outputs. It could classify all the strains in their species group. This network completes a chemotaxonomic method for Corynebacterium identification.

Postoperative endophthalmitis following intraocular lens (IOL) implantation is still one of the most feared complications of cataract surgery. Bacterial adhesion to IOLs during their insertion is a prominent etiological factor. Polypropylene was the first biomaterial that allowed this relation of cause and effect to be proven. Following adhesion, bacteria replicate, congregate and form multiple layers of microcolonies which actually represent the basic structural unit of the biofilm. The bacteria are embedded in a slime layer. Personal photographs illustrate the different steps of biofilm formation. This slime matrix is not only an adhesive medium; it also affects virulence. Adhesion to IOLs has been studied by several in vitro studies and discrepancies can be found between them which are due to variations of experimental conditions. The strains, the incubation times and the methods all varied. Adhesion is affected by the nature of the IOLs, the isolates and the surrounding medium. Since this medium is very difficult to model because of its complexity, in vivo studies seemed essential. We have recently determined in vivo evolution of the amount of attached bacteria to five types of IOLs. Crystalline lenses from 90 domestic pigs were removed aseptically and replaced with previously infected IOLs. There have been few epidemiological studies published to determine the relationship between endophthalmitis and the IOL type. However, the perfect biomaterial that could prevent postoperative endophthalmitis does not yet exist. Globally, hydrophilic materials and hydrophobic acrylic seem to be less sticky than silicone or PMMA, but this remains to be proven clinically.

Purpose: To analyze and compare the adherence of Staphylococcus epidermidis to intraocular lenses (IOLs) made of five different biomaterials (native or heparinized polymethylmethacrylate, silicone, hydrophilic acrylic, or hydrogel) and to detail the different steps and mechanisms of bacterial adhesion to a polymer., Methods: A clinical strain carrying the intercellular adhesion (ica) locus was used. In a previous study, the extent of bacterial binding was measured by counting. In this study, two different techniques, bioluminescence and scanning electron microscopy (SEM), were used to analyze the accuracy of each one, to obtain a comparison between the various IOLs, and to complete previous observations. The results were compared using both the Kruskal-Wallis and the Mann-Whitney nonparametric tests., Results: Bacterial adhesion was statistically weakest on hydrogel and then on hydrophilic acrylic polymer. Adhesion depended on the hydrophobicity or hydrophilicity of the biomaterials. Slight differences were found between the two methods, and these differences are explained. Furthermore, SEM observations highlighted two different patterns of bacterial adhesion (isolated bacteria and clusters of bacteria), assuming that hydrophobic IOLs (silicone and PMMA) probably facilitate bacterial colonization and biofilm production., Conclusions: Attachment mechanisms may be different in each case, depending on the polymer material and the infecting organism, because there are various types of behavior among S. epidermidis strains. Hydrophilic polymer surfaces (hydrogel and probably hydrophilic acrylic) seem to be useful in avoiding the development of bacterial colonies and hence in preventing endophthalmitis. Fewer bacteria were attached, demonstrating inhibition or delay in bacterial colonization.

Purpose: To determine whether the Staphylococcus epidermidis strain carries the intercellular adhesion (ica) locus, which encodes production of adhesins mediating adherence to biomaterials and to study, with scanning electron microscopy, the morphologic features of this coagulase-negative Staphylococcus strain that adheres to intraocular lenses (IOLs)., Methods: Polymerase chain reaction amplification was used to investigate whether the isolate under study (S. epidermidis clinical strain N890074) carries the ica locus. Sterile intraocular lenses (IOLs) were incubated in bacterial suspension either for 5 minutes or 1 hour. IOLs were then examined by scanning electron microscopy., Results: Polymerase chain reaction amplification revealed that S. epidermidis N890074 contained the ica locus. The bacteria appeared to be anchored to the surface of the lenses by several different means-particularly by leglike appendages and a slime layer-which probably came into play step by step., Conclusions: For the first time in ophthalmology, to the authors' knowledge, photographs showing leglike appendages involved in the first phase of adhesion have been obtained. They also clearly visualize the slime layer containing the embedded bacteria. This study provides information about the nature and the genesis of these attachment processes. Adherence is known to be greater when the bacterial DNA contain the ica locus. Full knowledge of the pathogenesis of bacterial adhesion is necessary to gain a better understanding of IOL infection and endophthalmitis.

Mitis group streptococci are pioneer colonizers of tooth surfaces and are implicated in various pathologies. Thus, accurate identification of oral mitis group strains would be valuable for studies of plaque ecology and dental caries and for diagnostic use in endocarditis or sepsis patients. The aim of this study was to evaluate the usefulness of PCR-RFLP analysis of the 16S-23S intergenic spacer for differentiating and identifying streptococcus mitis group species. The 16S-23S rDNA spacer regions of 27 type and reference Streptococcus strains, representing 8 species, were studied by PCR-mediated amplification by using oligonucleotide primers FGPS 1490-72 and FGPL 132'-38. PCR products were digested, independently, with 14 restriction enzymes. Only AluI, MboI, CfoI, HinfI and MaeII distinguished some species, particularly AluI and CfoI, but not all the species. Eight clusters were clearly generated, corresponding to currently recognized species, but only with the addition of five ITS restriction patterns, generated by AluI + MboI + CfoI + HinfI + MaeII, then clustered by UPGMA, on a distance consensus matrix. The combination of these five ITS RFLP tests allowed a relatively conclusive genomic group differentiation of mitis group species. Despite this observation, more strains of each species will need to be analyzed, particularly clinical isolates, before arriving at general conclusions about the utility of ITS restrictions for identification of strains at the species level. An ITS PCR-RFLP-based identifying method for streptococcus mitis group species would provide significant advantages over other molecular taxonomic methods which require DNA extraction and DNA-DNA hybridization.

We report here the application of pyrolysis-gas chromatography followed by atomic emission detection (AED) for the characterisation of Corynebacterium amycolatum and related species (i.e., C. striatum, C. minutissimum, C. xerosis and the recently described C. freneyi). This phenotypic method, which analyses the whole chemical composition of bacteria, clearly separates C. amycolatum from other species. Moreover, this C. amycolatum group is subdivided into two distinct subgroups. We cannot differentiate the C. minutissimum strains from those of C. striatum. On the other hand, C. freneyi and C. xerosis are clearly distinct from the other species.

The aim of this study was to determine the adherence of Staphylococcus epidermidis to intraocular lenses made of five different biomaterials: polymethylmethacrylate (PMMA), heparinized PMMA, silicone, hydrophilic acrylic, and hydrogel. The extent of bacterial binding was measured by counting. The results were compared using a one-factor variance analysis. Adherence was weakest on hydrogel and strongest on the silicone polymer. Bacterial adherence to the implant surface must therefore depend on the hydrophobicity or hydrophilicity of the biomaterial.

Three coryneform strains from clinical specimens were studied. They belonged to the genus Corynebacterium, since they had type IV cell walls containing corynemycolic acids. They had phenotypic characteristics that included alpha-glucosidase, pyrazinamidase and alkaline phosphatase activities and fermentation of glucose, ribose, maltose and sucrose. These are the characteristics of Corynebacterium xerosis. Since this species is very rare in human pathology, the strains were studied in more detail by comparing the 16S-23S intergenic spacers, rDNA sequences and levels of DNA similarity of these three strains and those of the reference strains C. xerosis ATCC 373T and Corynebacterium amycolatum CIP 103452T. According to DNA-DNA hybridization data, the three novel strains are members of the same species (level of DNA similarity >72%). Phylogenetic analysis revealed that these strains are closely related to C. xerosis and C. amycolatum, but DNA-relatedness experiments showed clearly that they constitute a distinct new species, with levels of DNA relatedness of less than 23% to C. xerosis ATCC 373T and less than 5% to C. amycolatum CIP 103452T. Two other alpha-glucosidase-positive strains presenting the same biochemical characteristics were included in the study and proved to be C. amycolatum. This new species can be differentiated from C. xerosis and C. amycolatum strains by carbon source utilization, intergenic spacer region length profiles and some biochemical characteristics such as glucose fermentation at 42 degrees C and growth at 20 degrees C. The name Corynebacterium freneyi sp. nov. is proposed with the type strain ISPB 6695110T (= CIP 106767T = DSM 44506T).

We report here the application of pyrolysis-gas chromatography followed by atomic emission detection (AED) for the characterisation of microorganisms. AED measured the quantity of carbon, sulfur and nitrogen in the molecules separated chromatographically. Twenty-three strains, representing eight Corynebacterium species, were tested in this preliminary study. Co-ordinate principal analysis grouped 11 strains in their respective species group. Most of the other strains appear randomly distributed, perhaps because these strains require additional nutrients. These preliminary results show that the method could be used as a tool for the taxonomic and perhaps the epidemiologic characterisation of bacteria.

We tested the carbon substrate assimilation patterns of 40 Corynebacterium amycolatum strains, 19 C. minutissimum strains, 50 C. striatum strains, and 1 C. xerosis strain with the Biotype 100 system (bioMérieux, Marcy-l'Etoile, France). Twelve carbon substrates of 99 allowed discrimination among the species tested. Additionally, assimilation of 3 of these 12 carbon substrates (maltose, N-acetyl-D-glucosamine, and phenylacetate) was tested with the API 20 NE identification system (bioMérieux). Since concordant results were observed with the two systems for these three carbon substrates, either identification system can be used as a supplementary tool to achieve phenotypic differential identification of C. amycolatum, C. minutissimum, and C. striatum in the clinical microbiology laboratory.

In a multicenter study, 407 strains of coryneform bacteria were tested with the updated and extended API (RAPID) Coryne system with database 2.0 (bioMérieux, La-Balme-les-Grottes, France) in order to evaluate the system's capability of identifying these bacteria. The design of the system was exactly the same as for the previous API (RAPID) Coryne strip with database 1.0, i.e., the 20 biochemical reactions covered were identical, but database 2.0 included both more taxa and additional differential tests. Three hundred ninety strains tested belonged to the 49 taxa covered by database 2.0, and 17 strains belonged to taxa not covered. Overall, the system correctly identified 90.5% of the strains belonging to taxa included, with additional tests needed for correct identification for 55.1% of all strains tested. Only 5.6% of all strains were not identified, and 3.8% were misidentified. Identification problems were observed in particular for Corynebacterium coyleae, Propionibacterium acnes, and Aureobacterium spp. The numerical profiles and corresponding identification results for the taxa not covered by the new database 2.0 were also given. In comparison to the results from published previous evaluations of the API (RAPID) Coryne database 1.0, more additional tests had to be performed with version 2.0 in order to completely identify the strains. This was the result of current changes in taxonomy and to provide for organisms described since the appearance of version 1.0. We conclude that the new API (RAPID) Coryne system 2.0 is a useful tool for identifying the diverse group of coryneform bacteria encountered in the routine clinical laboratory.

We studied two coryneform strains from clinical specimens. These strains had type IV and corynemycolic acids in their cell walls and also had phenotypic characteristics, such as urease activity and fermentation of glucose and sucrose but not trehalose, which did not permit assignment to any previously recognized taxon. According to DNA-DNA hybridization data, these two strains are members of the same species (level of DNA similarity, 86%). Phylogenetic analysis based on comparisons of almost complete small-subunit ribosomal DNA sequences revealed that these strains are closely related to Corynebacterium minutissimum, but DNA relatedness experiments clearly showed that they constitute a distinct new species with a level of DNA relatedness to the C. minutissimum type strain of less than 40%. This new species can be differentiated from C. minutissimum strains by its enzymatic activities and carbon source utilization, and the name Corynebacterium singulare is proposed for it. The type strain is strain IBS B52218 (= CCUG 37330), which was isolated from a semen specimen.

Turicella otitidis is a newly described coryneform bacterium isolated from middle ear fluids. We report here on the diagnosis of a strain isolated from otorrhea. The API Coryne system (bioMérieux, Marcy I'Etoile, France) used alone failed to differentiate T. otitidis, Corynebacterium afermentans, and Corynebacterium auris (ANF group). Biochemical tests such as DNase, enzymatic reactions (API ZYM; bioMérieux), and carbon substrate assimilation tests (Biotype 100; bioMérieux) allow presumptive identification. However, only chemotaxonomy and molecular biology can achieve unequivocal differentiation among these three species.