Escherichia coli spheroplasts in a Croatian patient misclassified by two urine sediment analysers as erythrocytes: case report.

INTRODUCTION: It has already been reported that subinhibitory concentrations of ß-lactam antibiotics can cause abnormal changes of bacterial forms, such as spheroplasts. Herein we report a case of Croatian male patient with Escherichia coli spheroplasts present in urine after treatment with tazobactam, on the tenth day of hospitalization. The aim of this report is to emphasize the inability of imaging based automated urine analysers to recognize some relatively uncommon forms of bacterial presentation in urine sediment. MATERIALS AND METHODS: During routine urine analysis, unusual particles were observed in patient urine. Urine sediment was examined by two urine analysers: Atellica 1500 (Siemens, Germany) and Iris iQ200 (Beckman Coulter, USA). Additionally, urine was sent for culture testing to Microbiology department. RESULTS: Both urine analysers didn't indicate presence of bacteria in urine sediment. Unusual particles observed on the tenth day were classified as erythrocytes by both instruments. Dipstick test showed blood trace and microscopic analysis revealed bacteria in urine. Urine culture was positive for Escherichia coli. Careful examination of urine sediment has confirmed that shapes present in urine were abnormal bacterial forms called spheroplasts. CONCLUSIONS: Imaging based automated urine analysers are not able to recognize bacterial spheroplasts in urine sediment misclassifying it as erythrocytes. Microscopic examination remains the gold standard for urines with blood trace or negative blood, in which erythrocytes are reported by urine analyser in urine sediment. Failure to identify and follow up such cases may lead to inaccurate treatment decisions and puts patient safety at risk.

Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model.

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml-1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria.IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

Probing Inner Membrane Protein Topology by Proteolysis.

Inner membrane proteins are inserted into the membrane via α-helices. These helices do not only constitute membrane anchors but may mediate specific interactions with membrane protein partners or participate in energetic processes. The number, location, and orientation of these helices is referred to as topology. Bitopic membrane proteins that consist of a single membrane-embedded domain connecting two soluble domains are distinguished from polytopic ones that consist of multiple membrane-spanning helices connected by extramembrane domains. Defining inner membrane protein topology could be achieved by different methods. Here we describe a protease accessibility assay that makes it possible to define topology based on digestion profiles.

Linalool isomerase, a membrane-anchored enzyme in the anaerobic monoterpene degradation in Thauera linaloolentis 47Lol.

BACKGROUND: Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Δ(1)-Δ(2)-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool. RESULTS: The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42 ± 0.28 nkat mg * protein(-1) and a kM value of 455 ± 124 µM were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 °C. CONCLUSION: The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers.

Phaeospirillum oryzae sp. nov., a spheroplast-forming, phototrophic alphaproteobacterium from a paddy soil.

Two strains (JA317(T) and JA559) of spiral shaped, spheroplast-forming, anaerobic, gram-negative, motile purple non-sulfur bacteria were isolated from rhizosphere soils of paddy and were characterized by a polyphasic taxonomic approach. Bacteriochlorophyll a and carotenoids, rhodopin, lycopene and rhodopin glucoside, were present as photosynthetic pigments. Intracellular photosynthetic membranes were of stacked type. The major fatty acids were C(18 : 1)ω7c, C(16 : 0) and C(16 : 1)ω6c/C(16 : 1)ω7c in both strains. The genomic DNA G+C content was 63.3±0.8 mol%. The two strains were closely related (mean DNA-DNA hybridization >85 %). Phylogenetic analysis showed that the strains clustered with the species of the genus Phaeospirillum, which belongs to the family Rhodospirillaceae within the class Alphaproteobacteria. Based on 16S rRNA gene sequence analysis, strains JA317(T) and JA559 showed highest sequence similarity with the type strains of Phaeospirillum chandramohanii (98.2 %), Phaeospirillum molischianum (97.4 %) and Phaeospirillum fulvum (97.1 %) of the family Rhodospirillaceae. Strain JA317(T) can be clearly distinguished from P. chandramohanii with respect to spheroplast formation and several other morphological and physiological properties. DNA-DNA relatedness of strain JA317(T) with its closest relatives of the genus Phaeospirillum was less than 55 %. It is evident from the phenotypic, chemotaxonomic and molecular genetic evidence that strain JA317(T) represents a novel species of the genus Phaeospirillum, for which the name Phaeospirillum oryzae sp. nov., is proposed. The type strain of the species is JA317(T) ( = NBRC 104938(T)  = KCTC 5704(T)).

Novel method for preparing spheroplasts from cells with an internal cellulosic cell wall.

Protoplast and spheroplast preparations allow the transfer of macromolecules into cells and provide the basis for the generation of engineered organisms. Crypthecodinium cohnii cells harvested from polyethylene glycol-containing agar plates possessed significantly lower levels of cellulose in their cortical layers, which facilitated the delivery of fluorescence-labeled oligonucleotides into these cells.

Preparation of protein extracts from yeast.

Three protocols are presented for preparing protein extracts; they differ primarily in the way the cells are broken. In the basic protocol, cells are enzymatically converted to spheroplasts, which are then lysed by a combination of osmotic shock and Dounce homogenization. A support protocol for isolating intact nuclei by differential centrifugation is also presented. An alternate protocol describes mechanical breakage of cells by vortexing in the presence of glass beads. In a second alternate protocol, growing cells are frozen immediately in liquid nitrogen and then lysed by grinding in an industrial-strength blender in the presence of liquid nitrogen.

[The ultrastructural characteristics of Neisseria meningitidis strains isolated in different forms of infection]. / Osobennosti ul'trastruktury shtammov Neisseria meningitidis, vydelennykh pri razlichnykh formakh infektsii.

The electron microscopic study of cell populations of the first generation of several N.meningitidis strains, isolated from humans and grown on artificial culture medium (Hottinger's serum agar) has made it possible to establish that these organisms are morphologically heterogeneous. It was manifested by the presence of 7 morphological variants of cells in their cultures (different strains have different proportions of these variants): (a) "normal" and similar to normal diplococcal variants; (b) atypically dividing cells; (c) spheroplastic forms; (d) protoplastic forms (typical and atypical); (e) variably gemmating forms; (f) microforms and (g) gigantic forms. These data are of interest for the microbiological diagnosis of meningitis.

Identification of spheroplast-like agents isolated from tissues of patients with Crohn's disease and control tissues by polymerase chain reaction.

Mycobacterium paratuberculosis has been isolated from tissue taken from patients with Crohn's disease and has been implicated in the etiology of this disease. On culture, the organisms appear initially as cell wall-deficient, spheroplast-like forms that are difficult to identify by conventional techniques. Here we examine 30 unidentified cultures by the polymerase chain reaction using primers specific for M. paratuberculosis, Mycobacterium tuberculosis, and Mycobacterium avium restriction fragment length polymorphism type A/I and also by a non-species-specific mycobacterial polymerase chain reaction. Six of these cultures, all from Crohn's disease, were shown to contain DNA from M. paratuberculosis. Cultures from both Crohn's disease and controls were found to contain mycobacterial DNA of unknown specific origin.

A fast spheroplast formation procedure in some 2,5-diketo-D-gluconate- and 2-keto-L-gulonate- producing bacteria.

Calcium 2-keto-L-gulonate (Ca-2-KLG, a key intermediate in vitamin C synthesis) is produced from calcium 2,5-diketo D-gluconate (Ca-2,5-DKG) by a variety of bacteria. A few bacterial species which efficiently convert glucose to Ca-2,5-DKG have been isolated in our laboratory. Our bacterial collection included species that possess the genes for production of Ca-2-KLG from Ca-2,5-DKG; however, the yield of the former is poor. A procedure for the preparation of spheroplasts in Ca-2,5-DKG- and Ca-2-KLG-producing bacteria was developed for the construction of recombinants (fusants), combining the genes for conversion of glucose to Ca-2-KLG efficiently by protoplast fusion. The standard procedure for spheroplast formation in Gram negative bacteria by the Tris-sucrose-EDTA-lysozyme system did not work in the organisms under investigation. The need for an alternative method was necessary. Our results show that, while the Tris-NaCl-EDTA-lysozyme system (pH 8.3) worked very well with bacterial strains of Gluconobacter oxydans (ATCC9937) and Acetobacter melanogenus (NCIM2259), the Tris-sucrose-EDTA-lysozyme system worked well for Erwinia herbicola (ATCC21998), Pseudomonas chlororaphis (NCIM2041) and Corynebacterium species (ATCC31090). However, none of these systems produced spheroplasts in Brevibacterium ketosoreductum (ATCC21914), for which a separate system is under development.

Isolation of DNA from yeasts.

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.

[Isolation and ultrastructure of Yersinia spheroplasts and protoplasts]. / Poluchenie i ul'trastruktura sferoplastov i protoplastov u iersinii.

The conditions suitable for the cultivation of Yersinia and the inhibition of their cell-wall synthesis have been selected with the aim of obtaining spheroplasts and protoplasts of these microorganisms. Penicillin, streptomycin and lithium chloride have proved to be not very suitable for this purpose as they induce essential changes in the structure of the cytoplasmic membrane in altered Yersinia forms. The addition of 1% of glycine (for Y. pseudotuberculosis), 1-1.5% of methionine in combination with growth stimulators has made it possible to obtain Yersinia spheroplasts and protoplasts with the intact cytoplasmic membrane, thus permitting the isolation and purification of the cytoplasmic membrane fraction.

Progress in culture and subculture of spheroplasts and fastidious acid-fast bacilli isolated from intestinal tissues.

The efficiency of culture media was compared for the culture and subculture of very slowly growing acid-fast bacilli and spheroplast forms obtained from intestinal tissues of patients with Crohn's disease and ulcerative colitis and from controls without inflammatory bowel disease. Media were developed by modifying a nutrient broth medium based on veal infusion broth and yeast extract. We evaluated the effects of pH and the addition of Tween 80, Dubo oleic albumin complex, an extract from intestinal tissue from a patient with Crohn's disease, horse serum, sucrose, magnesium sulfate, ferrous ammonium sulfate, and sodium citrate. All media contained mycobactin J (2 micrograms/ml). We developed a medium (MG3) which was highly successful in promoting the growth of very fastidious organisms and promoted reversion of spheroplasts to acid-fast rods. MG3 contained veal infusion broth, 1% yeast extract, 10% horse serum, 0.3 M sucrose, 0.2% MgSO4, 0.1% ferrous ammonium sulfate, 0.1% sodium citrate, and 2 mg of mycobactin J per liter. We were able to obtain quantities of organisms sufficient for examination of the organisms by molecular techniques. Successful cultivation of all isolates and reversion of spheroplasts to acid-fast forms encourage further studies of the possibility of a complex association of mycobacteria and Crohn's disease.

[Isolation of spheroplasts from Escherichia coli 85 for aspartate-ammonia-lyase localization]. / Poluchenie sferoplastov iz Escherichia coli 85 dlia opredeleniia lokalizatsii spartat-ammiak-liazy.

Conditions were determined for preparation of spheroplasts from E. coli under the action of lysozyme in the presence of EDTA. The preparation took from 10 to 15 min. The degree of conversion to spheroplasts was monitored spectrophotometrically at 660 nm. The spheroplasts formed were unstable in Tris-HCl buffer and immediately lysed, but they were more stable in 1 M sucrose. At lysozyme concentrations above 40 micrograms/ml of the reaction mixture, the cells lysed to a greater extent. The distribution of aspartate ammonia-lyase activity between the precipitate of the spheroplasts and the supernatant allowed the authors to suggest that aspartase should be located in the cytoplasm.

Biochemical studies on a factor isolated from Klebsiella K43-BTS1 that cross-reacts with cells from HLA-B27 positive patients with ankylosing spondylitis.

A component of the cell walls of certain enteric bacteria has been identified that cross-reacts with an HLA-B27-associated cell-surface structure on lymphocytes and other cell types from patients with ankylosing spondylitis. This component, or "modifying factor," from one particular organism, Klebsiella K43-BTS1, has been studied in detail. A purification scheme has been developed using preparative electrofocusing and gel-permeation high performance liquid chromatography techniques and the purified material used in various characterization studies. A previous study demonstrated that the modifying factor has an approximate molecular weight of 30,000 and an isoelectric point of 5.4-5.5. In this study two-dimensional gel electrophoresis experiments demonstrated that the modifying factor is associated with a single protein component of the cell wall of this organism. Pronase and papain destroyed the modifying factor activity whereas trypsin and alpha-chymotrypsin degraded the factor into smaller fragments without destroying its ability to modify B27+ AS- lymphocytes. Neuraminidase did not affect the modifying factor itself but did affect B27+ AS- lymphocytes such that they became unresponsive to modification. Sugar inhibition studies suggested that sugar groups are probably not involved in the function of the modifying factor. The availability of purified modifying factor should permit more detailed chemical analyses as well as functional studies to determine the significance of this molecule to the pathogenesis of ankylosing spondylitis.

Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease.

Two strains of an unclassified Mycobacterium species were isolated after 18 and 30 months of incubation of media inoculated with resected intestinal tissues from patients with Crohn's disease. These strains represented the third and fourth isolates of this organism from Crohn's disease patients. Ultrastructural examination of this strain and two previously isolated strains revealed the presence of spheroplasts which eventually transformed into the bacillary form of a previously unrecognized Mycobacterium species. These cell wall-deficient forms did not stain with conventional dyes and failed to grow on hypertonic media. Restriction polymorphism of the ribosomal DNA genes was used to determine the relationship between the cell wall-deficient and bacillary forms. Identical restriction patterns of the ribosomal DNA genes were found between the spheroplasts and Mycobacterium sp. isolates with EcoRI, BamHI, and XhoI restriction endonucleases, thus providing definitive evidence of their origin. Unidentified spheroplasts were isolated from an additional 12 patients with Crohn's disease, of which 7 of 10 seroagglutinated with antiserum prepared against the Mycobacterium sp. Spheroplasts were isolated from 16 of 26 (61%) patients with Crohn's disease but not from tissues of 13 patients with ulcerative colitis or 13 patients with other diseases of the bowel. These findings support the role of mycobacteria as etiologic agents in some cases of Crohn's disease.

[Validation and development of an immunofluorescence method for isolating and identifying mycobacteria L forms]. / Obosnovanie i razrabotka vyiavleniia i identifikatsii L-form mikobakterii metodom immunofliuorestsentsii.

The complement fixation test and the immunofluorescence test have demonstrated that the L-forms of mycobacteria retain their species-specific and genus-specific determinants and possess serological activity. The L-variants obtained by different methods differ in size, depending on the degree of the destruction of their cell wall. Specific antisera to the L-forms of mycobacteria, suitable for use in the indirect immunofluorescence test, have been obtained. These antisera are highly specific and permit not only the rapid detection, but also identification of the L-forms.