Abstract: 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.
Abstract: A novel strain, C-138(T), belonging to the genus Corynebacterium was isolated from a severe thigh liposarcoma infection and its differentiation from Corynebacterium xerosis and Corynebacterium freneyi is described. Analysis of 16S rRNA gene sequences, rpoB sequences and the PCR profile of the 16S-23S spacer regions was not conclusive enough to differentiate strain C-138(T) from C. xerosis and C. freneyi. However, according to DNA-DNA hybridization data, strain C-138(T) constitutes a member of a distinct novel species. It can be differentiated from strains of C. xerosis and C. freneyi by colony morphology, the absence of alpha-glucosidase and some biochemical characteristics such as glucose fermentation at 42 degrees C and carbon assimilation substrates. The name Corynebacterium hansenii sp. nov. is proposed for this novel species; the type strain is C-138(T) (=CIP 108444(T)=CCUG 53252(T)).
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Most currently used disinfectants for dialysis machines have a good bactericidal efficacy on biofilms but leave dead cells on the surface. This contributes to the regrowth of biofilm and the release of pyrogens. A new anti-biofilm procedure consisting of sequential treatment combining enzymes and detergents is able to detach adherent cells. The efficacy of this procedure was assessed both in vitro and in reality. For in vitro studies, a biofilm model was set up. Studies were also performed in reality in a clinically used dialysis machine. Biofilm removal was first monitored by image analysis. Then, the biomass was detached by scraping and quantified by plate counts and endotoxin level measurement. Treated samples were compared to untreated control samples. The procedure led to the complete detachment of the biomass, both in vitro and in the reality situation. The aim of this procedure is to replace or complete the usual disinfection methods for medical devices.
Abstract: We conducted an in vitro study to investigate the antibacterial activity of clonidine and neostigmine on common microorganisms encountered during infectious complications after regional anesthesia. Standardized suspensions of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli were incubated during 1, 3, 6, and 24 h at 37 degrees C with concentrations of 37.5, 75, and 150 microg/mL of clonidine and 125, 250, and 500 microg/mL of neostigmine. After 24 h incubation at 37 degrees C, the colony counts were compared by two-way analysis of variance. The mean colony counts for S. aureus decreased significantly from control as the exposure to clonidine increased (P < 0.05), with a approximately 100% kill at 6 h for the largest concentration (150 microg/mL) and at 24 h for the intermediate concentration (75 microg/mL). Similar results were observed for S. epidermidis, with a approximately 100% kill at 6 h for the largest concentrations (75 and 150 microg/mL). No bactericidal activity of clonidine was observed for E. coli and no bactericidal activity of neostigmine was observed for any of the tested strains. In the conditions of this experiment, clonidine, but not neostigmine, exhibited a concentration-dependent and time-dependent bactericidal activity in vitro on the microorganisms most frequently encountered in infectious complications after regional anesthesia.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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).
Abstract: 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.
Abstract: 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.
Abstract: The phenotypic (antibiotype, serotype, phagetype) and genotypic (SmaI restriction patterns using pulsed-field gel electrophoresis) characters of 162 Staphylococcus aureus epidemiologically unrelated strains were studied. Eighty-two of the isolates produced enterotoxin-A (SEA+), while 80 produced none (SEA-). None of the phenotypic characters observed were characteristic of SEA+ strains. On the other hand, the electrophoretic profiles revealed a non-random distribution of the SEA+ strains (p < 0.01 in groups PI and PIII, and p < 0.03 in group PII). It can therefore reasonably be assumed that the enterotoxin-A-producing strains did not constitute a single clone, but rather, seemed to belong to strains derived from at least three clones with distinct genetic organization.
Abstract: Sixty-nine Staphylococcus aureus strains, 39 of which produced staphylococcal enterotoxin B (SEB+) and 14 of which were associated with toxic shock (TS+), were studied using the following markers: serotyping, phage typing, antibiotyping, ribotyping, zymotyping and pulsed-field electrophoresis typing. Analysis of the results showed that the enterotoxin B producing strains were derived from at least three clones: the first two consisted of methicillin-susceptible strains, while the third included the methicillin-resistant (MRSA) strains. TS+ strains of nongenital origin appeared to be distributed between the three clones, with no specific characters.
Abstract: In order to recognize particular characteristics of pathogenic strains, epidemiologic markers of 27 Staphylococcus epidermidis strains (9 pathological and 18 commensal) were studied. Nine strains were responsible of infective endocarditis (8 on native valves, and 1 on prosthetic valve). No case occurred after admission to hospital or surgery. Eighteen commensal strains were isolated from control subjects who had had no contact with the hospital environment and who had not received a recent antibiotic treatment. The microbiological characteristics were so diverse that no differentiation between the pathogenic strains and commensal strains could be done and no particular pathogenic clone was recognized.
Abstract: A recent outbreak of erythroderma in young children in an Albanian hospital was investigated. The etiology was not established, but Staphylococcus haemolyticus was frequently isolated from the affected children and from staff working in the same unit. Possible relationships among the isolates were investigated by using classical techniques (biotype, antimicrobial susceptibility, and extrachromosomal DNA pattern) and by restriction endonuclease analysis (REA) of total DNA. Control isolates of proven pathogenicity from hospitalized patients in Lyon, France were subjected to the same procedures. Distinct REA patterns were obtained after digestion with two enzymes in 7 of 10 isolates from five affected children. Six distinct patterns were observed in nine isolates from six staff members; two REA patterns from patient isolates and two from staff members were identical, and these were distinguishable by the other markers examined. Only two different REA patterns were found in the pathogenic control isolates despite the use of a third additional enzyme. Again, the isolates with the same REA patterns could be distinguished by their plasmid profile or antimicrobial resistance profile. REA of total DNA used in combination with other markers indicated that the Albanian isolates differed considerably, whereas the French pathogenic isolates showed little variability.
Abstract: The cellular fatty acids of 39 strains belonging to the genus Aeromonas (Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Aeromonas media, Aeromonas schubertii, Aeromonas veronii) were determined by high resolution gas-liquid chromatography. The fatty acid profiles were characterized by major amounts (60% or more) of one saturated (hexadecanoic acid = 16:0) and two unsaturated (hexadecenoic acid = 16:1 and octadecenoic acid = 18:1) acids. While the majority of the strains of the six species exhibited, qualitatively, very similar fatty acid compositions, only minor and inconsistent differences could be observed which would be useful for a distinction of the different taxons. The following fatty acids were qualitatively identified: 12:0, i-13:0, 14:0, 3-OH 13:0, i-15:0, 15:0, 2-OH 14:0, 3-OH 14:0, i-16:0, 16:1, 16:0, i-17:1, i-17:0, a-17:0, 17:0 cyclopropane, 17:1, 17:0, 18:1 (3 isomers), 18:0 and i-20:0. Excellent congruence was found in reproducibility studies. Fatty acid analyses show a great homogeneity within the group and the technique does not appear to be the ideal method in distinguishing between Aeromonas species.
Abstract: The API 20 EC and ATB 32 GN identification systems were compared for their ability to identify 231 coliform bacteria strains. Agreement with the identification given by conventional methods was achieved for 96.1 p. cent of strains by the API 20 EC gallery and for 95.9 p. cent by the ATB 32 GN system. Complementary tests were needed to identify 9.5 p. cent of strains using the API 20 EC system but 30.3 per cent using the ATB 32 GN system.
Abstract: Variation in typing of clinically significant isolates of coagulase-negative staphylococci (CNS) was determined by five typing methods with 143 isolates obtained from 19 patients over periods from 2 days to 1 year. In only one case did all isolates give exactly the same typing pattern by all five tests. No single method, or simple combination, provided a ready means of confirming the relatedness of separate isolates. The most frequently useful tests were antibiotic susceptibility and extrachromosomal DNA banding patterns. However, the results of biotyping, serotyping and phage typing were also helpful in showing the relationship between different isolates from a given patient. In most cases a core pattern varying by the gain or loss of a small number of features, characterised a given patient's isolates. In two causes, apparently radical changes in the infecting organism were observed, and confirmed by restriction endonuclease analysis. Care should be taken when successive isolates of CNS show distinct typing differences in deciding their clinical relevance.
Abstract: Extrachromosomal DNA analysis and restriction endonuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.
Abstract: We compared the epidemiological markers of 13 Staphylococcus epidermidis strains isolated from an adult inpatient during a febrile episode and 23 S. epidermidis strains isolated during a presumptive outbreak of nosocomial infection in a neonatal ward. The total DNA restriction endonuclease analysis (REA) was processed along with the following conventional markers: biotyping, serotyping, phage typing, antibiotic susceptibility profiles, and plasmid profiles. The REA method was reproducible, giving stable results both in vitro and in vivo. For the hospitalized adult patient, the conventional markers of the 13 strains were concordant and the restriction profiles were identical. Five restriction groups were demonstrated during the course of the outbreak. Within two of the groups, the identities of all of the markers were used to verify whether all of the isolates belonged to the same cell clone. In a third group, combined analysis of the conventional markers and REA had to be used to demonstrate isolate similarity. On the other hand, in another group, none of the markers were similar; interpretation was not easy. An epidemiological study of S. epidermidis infections in hospitals must take into account all of the epidemiological markers: biotypes, serotypes, phage types, antibiograms, plasmid profiles, and REA.
Abstract: The assimilation of carbon substrates by 103 strains of Aeromonas of different origin identified by conventional methods was studied by means of a standardized micromethod containing 147 tests (API system). Six distinct groups could be recognized and the discriminating substrates were determined. 3 species of Aeromonas can be identified by means of conventional method: A. hydrophila, A. sobria and A. caviae. The method has a number of drawbacks: Some media are unreliable, others are difficult to read, strict preservation conditions are essential. The proposed micromethod for carbon substrate assimilation allows, in most cases, a simple separation of the 3 motile Aeromonas species.
Abstract: DNA could not be quickly extracted from members of the genus Actinomyces by the usual methods of lysis. Treatment of 7 different actinomyces cells with lysozyme and achromopeptidase, both 5 mg/g wet cells, for 2 h, followed by SDS (0.2%), proteinase K (5 mg/g wet cells) and EDTA (lmM) for 1 h, lysed the cells. The yield obtained in one day was 337 micrograms per 200 mg of bacterial cells. The treatment was also found to work effectively on strains belonging to Veillonella, Staphylococcus, Fusobacterium and Bifidobacterium genera.
Abstract: We report here the first sensitive enzyme immunoassay of a hapten. A progesterone beta galactosidase conjugate was prepared using carbodiimide as a bifunctional reagent. Rabbit progesterone antisera were previously obtained. The separation of the bound from the free fraction of the label was performed with the help of polymerized anti rabbit gamma-globulins. The enzyme activity of the bound fraction was determined with O-nitrophenyl-beta-D-galactoside as substrate. Specificity and sensitivity (approximately 15 pg) of this enzyme immunoassay can be successfully compared with radioimmunoassay performances. It thus provides a non radioactive, inexpensive and reliable method of small molecule quantitation.
