Rapid differentiation of cocci/mixed bacteria from rods in voided urine culture of women with uncomplicated urinary tract infections.

OBJECTIVES: To evaluate the ability of laser flow cytometry to predict cocci/mixed growth in the pre-analytical phase of urine specimens. METHODS: We retrospectively reviewed urine samples from women with uncomplicated urinary tract infections from urologic clinics for study. Urine analyses were performed with laser flow cytometry (UF1000i, Sysmex, Kobe, Japan) and then diagrams were generated (forward scatter vs. fluorescent light scatter). Each specimen (bacteria count >357 BACT/µL) was classified as either cocci bacteria or rods/mixed growth according to the diagrams. Standard urine cultures were performed, and the agreement between cultures and the UF1000i interpretations was analyzed with kappa statistics. RESULTS: Finally, 491 specimens met the criteria for analysis. Among the 376 specimens with single bacteria growth, there were 26 gram-positive cocci (13 Streptococci spp., 7 Staphylococci spp., 6 Enterococci spp.), 1 gram-positive rods (Corynebacterium spp.), and 349 gram-negative rods (273 Escherichia coli, 33 Klebsiella spp., 29 Proteus spp., 6 Citrobacter spp., 4 Enterobacter spp., 3 Pseudomonas spp., and 1 Providencia spp.). There were 115 specimens with two bacteria species or more that were regarded as mixed growth. Agreement of rods or cocci/mixed growth between the laser flow cytometry and urine cultures yielded a kappa value of 0.58. The positive and negative predictive rate of the UF1000i for cocci/mixed growth in voided urine culture was 81.8% and 84.7%, respectively. CONCLUSIONS: Through laser flow cytometry, we can predict growth of cocci/mixed growth in the pre-analytical phase of urine culture, thus avoiding unnecessary urine culture and waiting time.

How to get (a)round: mechanisms controlling growth and division of coccoid bacteria.

Bacteria come in a range of shapes, including round, rod-shaped, curved and spiral cells. This morphological diversity implies that different mechanisms exist to guide proper cell growth, division and chromosome segregation. Although the majority of studies on cell division have focused on rod-shaped cells, the development of new genetic and cell biology tools has provided mechanistic insight into the cell cycles of bacteria with different shapes, allowing us to appreciate the underlying molecular basis for their morphological diversity. In this Review, we discuss recent progress that has advanced our knowledge of the complex mechanisms for chromosome segregation and cell division in bacteria which have, deceptively, the simplest possible shape: the cocci.

Identification and classification of cocci bacterial cells in digital microscopic images.

In cytology, automating the feature extraction process yields an objective, quantitative, detailed and reproducible computation of cell morphofunctional characteristics and allows the analysis of a large quantity of images. The objective of the present study is to develop an automatic tool to identify and classify the different types of cocci bacterial cells in digital microscopic cell images. Geometric features are used to identify the arrangement of cocci bacterial cells, namely cocci, diplococci, streptococci, tetrad, sarcinae and staphylococci using 3σ, K-NN and Neural network classifiers. The current methods rely on the subjective reading of profiles by a human expert based on the various manual staining methods. In this paper, we propose a method for cocci bacterial cell classification by segmenting digital bacterial cell images and extracting geometric and statistical features for cell classification. The experimental results are compared with the manual results obtained by microbiology expert and other methods in the literature.

Bavariicoccus seileri gen. nov., sp. nov., isolated from the surface and smear water of German red smear soft cheese.

The phylogenetic position and physiological characters of six hitherto-unknown lactic acid bacterial isolates, which form part of the surface microbiota of German red smear soft cheese, are reported. The coccoid cells are aerotolerant, Gram-positive, catalase-negative and non-motile. The cell-wall peptidoglycan contains alanine, glutamic acid, lysine and aspartic acid and is of the A4alpha type (l-Lys-d-Asp). The sequences of the 16S rRNA, groEL and rpoB genes of the six isolates are identical and reveal that these isolates represent an independent lineage within the radiation of the family Enterococcaceae in the phylum Firmicutes. Their closest phylogenetic neighbour is the lactic acid bacterium Atopobacter phocae M1590/94/2(T), with which they share 94.9 % 16S rRNA gene sequence similarity; representatives of other genera such as Granulicatella, Carnobacterium and Trichococcus are more distantly related. DNA-DNA hybridization studies reveal that the six isolates are members of a single species, and this is confirmed by similarities in biochemical characteristics. The six isolates were assigned four different groups by Fourier-transform infrared and randomly amplified polymorphic DNA typing. Therefore, it is formally proposed that these isolates should be classified in a single novel species of a novel genus and be named Bavariicoccus seileri gen. nov., sp. nov. The type strain of Bavariicoccus seileri is WCC 4188(T) (=DSM 19936(T) =CCUG 55508(T)).

Characterisation of thermotolerant cocci from indigenous flora of 'leben' in algerian arid area and DNA identification of atypical Lactococcus lactis strains.

Lactic acid bacteria (LAB) are widely used in food industry and their growth performance is important for the quality of the fermented product. By combining results from conventional isolation methods and molecular investigation of 16S rRNA gene and lactococcal/enterococcal specific genes, we identify at species level catalase negative gram positive thermoresistant cocci isolated from traditional 'leben', a 24-h fermented milk in arid area of west Algeria. Forty strains phenotypically related to cocci LAB were identified as belonging to the species Lactococcus lactis ssp. lactis, Enterococcus faecalis, Enterococcus faecium, and other Enterococcus species. No Streptococcus thermophilus strain was isolated. Ten different phenotype groups were recognized, and the species content of these groups were in some cases different from the expected features usually given in genus and species descriptions. In particular, atypical lactococci, able to grow in 6.5% NaCl, at pH 9.5 and showing high resistance to thermal stresses were isolated. Lactococci, but also enterococci isolated from traditional 'leben' produced in the desert area, may be therefore of interest in milk fermentation. Further studies to assess this source of diversity within the wild microbial population should provide starter new strains for product innovation.

The different shapes of cocci.

The shape of bacteria is determined by their cell wall and can be very diverse. Even among genera with the suffix 'cocci', which are the focus of this review, different shapes exist. While staphylococci or Neisseria cells, for example, are truly round-shaped, streptococci, lactococci or enterococci have an ovoid shape. Interestingly, there seems to be a correlation between the shape of an organism and its set of penicillin-binding proteins--the enzymes that assemble the peptidoglycan, the main constituent of the cell wall. While only one peptidoglycan biosynthesis machinery seems to exist in staphylococci, two of these machineries are proposed to function in ovoid-shaped bacteria, reinforcing the intrinsic differences regarding the morphogenesis of different classes of cocci. The present review aims to integrate older ultra-structural data with recent localization studies, in order to clarify the relation between the mechanisms of cell wall synthesis and the determination of cell shape in various cocci.

Characterization of intact subcellular bodies in whole bacteria by cryo-electron tomography and spectroscopic imaging.

We illustrate the combined use of cryo-electron tomography and spectroscopic difference imaging in the study of subcellular structure and subcellular bodies in whole bacteria. We limited our goal and focus to bodies with a distinct elemental composition that was in a sufficiently high concentration to provide the necessary signal-to-noise level at the relatively large sample thicknesses of the intact cell. This combination proved very powerful, as demonstrated by the identification of a phosphorus-rich body in Caulobacter crescentus. We also confirmed the presence of a body rich in carbon, demonstrated that these two types of bodies are readily recognized and distinguished from each other, and provided, for the first time to our knowledge, structural information about them in their intact state. In addition, we also showed the presence of a similar type of phosphorus-rich body in Deinococcus grandis, a member of a completely unrelated bacteria genus. Cryo-electron microscopy and tomography allowed the study of the biogenesis and morphology of these bodies at resolutions better than 10 nm, whereas spectroscopic difference imaging provided a direct identification of their chemical composition.

Gram stain as a relapse predictor of bacterial vaginosis after metronidazole treatment.

Bacterial vaginosis is the most prevalent disease of the female genital tract. In spite of various effective antibiotics in the treatment of bacterial vaginosis, its high relapse rate is a common problem. Bacterial species causing bacterial vaginosis are generally unable to be cultured by conventional methods. It is impractical and inadequate to use culture methods to guide initial treatment. Gram stain of vaginal secretion is a practical tool to establish the diagnosis of bacterial vaginosis. We enrolled 78 cases of Gram stain-proven bacterial vaginosis and tried to use Gram stain as a predictor of relapse after 1 week of treatment with metronidazole. Possible predictive factors for relapse in Gram stain were analyzed, including absence of large Gram-positive rods, presence of small Gram-negative rods, small Gram-variable rods, curved Gram-variable rods, or Gram-positive cocci. Gram stain was repeated immediately after treatment, and at 1 month and 3 months after treatment. All cases showed beneficial clinical effect after metronidazole treatment. Eighteen cases (23.1%) relapsed during the follow-up period. All 16 cases with significant Gram-positive cocci in pretreatment smears relapsed after metronidazole treatment. Presence of small Gram-negative rods, small Gram-variable rods, and curved Gram-variable rods, or absence of large Gram-positive rods did not predict relapse. Gram-positive cocci in pretreatment smear was a good predictor of relapse after metronidazole treatment.

Psychrophilic Planococcus maitriensis sp.nov. from Antarctica.

An orange pigmented bacterium, S1, was isolated from a cyanobacterial mat sample collected in the vicinity of Schirmacher Oasis, Maitri, the Indian station, in Antarctica. The bacterium is Gram-positive and possesses all the characteristics of the genus Planococcus. It is non-sporulating, motile and has A4alpha type peptidoglycan, MK-7 and MK-8 as the major menaquinones and anteiso-C(15:0) as the major fatty acid. Based on the phylogenetic characteristics, the bacterium S1 is identified as a close relative of Planococcus citreus with which it shares 98.12% similarity at the 16S rRNA gene level but exhibits a low similarity of 52% at the whole genome level. Apart from the above major differences, S1 also exhibits phenotypic differences with Planococcus citreus and other members of the genus Planococcus. Based on these differences, the bacterium S1 is identified as a new species of the genus Planococcus for which the name Planococcus maitriensis is proposed. The type strain of Planococcus maitriensis is S1(T) (= MTCC 4827; DSM 15305).

Planococcus rifietensis sp. nov, isolated from algal mat collected from a sulfurous spring in Campania (Italy).

The taxomony of strain M8, isolated from algal mat formed at the origin of a sulfurous spring in Rifieto (Savignano Irpino, Campania, Italy), was investigated in a polyphasic approach. The morphological, physiological and genetic characteristics were compared with of Planococcus and Planomicrobium species. The isolate grew optimally at pH 9.0, 1.8 M NaCl at 37 degrees C. The cells were Gram-positive cocci that form pairs, tetrads and aggregates of several cells. The isolate was aerobic/microaerophilic and accumulated glycine-betaine, as a major osmolyte, with minor components glutamate and an unknown compound. M8 was able to hydrolyse X-Glc (5-bromo-4-chloro-3-indoyl beta-d-glucopyranoside). The polar lipid profile consisted of phosphatidylglycerol and diphosphatidylglycerol as major components, and phosphocholine as a minor compound. MK8 was the only quinone found and the fatty acid composition was dominated by branched acids, mainly aiC15:0. The G+C content of DNA was 47.9% and its phylogenetic position was established by 16S rRNA gene sequencing as a member of the genus Planococcus. The DNA/DNA similarity of M8 to the type species Planococcus citreus was less than 55%. For this reason and for physiological and chemotaxonomic features, it is proposed to create a new species Planococcus rifietensis sp. nov.

A multicenter evaluation of linezolid antimicrobial activity in North America.

Overall, 141 centers in North America enrolled in this international surveillance study designed to evaluate the in vitro antimicrobial activity and spectrum of linezolid, a new oxazolidinone. Each participant tested the susceptibility of clinical isolates of staphylococcal species (n = 85) against 12 drugs, and enterococcal species (n = 40) against 6 drugs using reference broth microdilution trays; and of streptococcal species (n = 25) against 6 drugs using Etests (AB BIODISK, Solna, Sweden). Quality control testing was conducted using recommended strains, and verification of resistance to linezolid and select other agents was performed by a regional monitor. Of the 20,161 isolates collected from sites across the United States (US; n = 132) and Canada (n = 9), 18,307 were included in this analysis. Oxacillin resistance occurred in 38.7 and 70.6% of Staphylococcus aureus and coagulase-negative staphylococcal (CoNS) isolates, respectively. Vancomycin resistance was reported in 65.9 and 2.6% of Enterococcus faecium and E. faecalis, respectively. Penicillin resistance occurred in 37.2% of Streptococcus pneumoniae, 17.5% constituting high-level resistance (MIC, > or =2 microg/ml). The MIC(90) for linezolid was 1 microg/ml for streptococci, 2 microg/ml for enterococci and CoNS isolates, and 4 microg/ml for S. aureus. Using the US FDA-recommended susceptible breakpoints for linezolid, there were no confirmed reports of linezolid resistance (i.e., MIC > or =8 microg/ml). The occurrence of linezolid MICs was unimodal and generally varied between, 1-4 microg/ml for staphylococci (94% of recorded results), 1-2 microg/ml for enterococci (93%), and 0.5-1 microg/ml for streptococci (85%). Susceptibility to linezolid was not influenced by susceptibility to other antiicrobials such as vancomycin, beta-lactams or macrolides. Only linezolid was universally active against essentially all tested Gram-positive specimens. The unimodal susceptibility pattern is indicative of excellent and near complete activity against key Gram-positive pathogens including multiply resistant strains, but surveillance for emerging resistances (rare) and the performance of routine susceptibility tests to guide patient therapy seems prudent.

Effect of the space environment on the induction of DNA-repair related proteins and recovery from radiation damage.

Recovery of bacterial cells from radiation damage and the effects of microgravity were examined in an STS-79 Shuttle/Mir Mission-4 experiment using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space-Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under microgravity. No difference was observed for the survival of radiosensitive mutant rec30 cells whether incubated in space or on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, the product of a newly found gene related to the DNA repair mechanism of D. radiodurans, was enhanced under microgravity compared with ground controls.

Facklamia tabacinasalis sp. nov., from powdered tobacco.

An unknown Gram-positive, catalase-negative, facultatively anaerobic, coccus-shaped organism originating as a contaminant of snuff was characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the bacterium represents a new subline within the genus Facklamia. The unknown bacterium was readily distinguished from Facklamis hominis and Facklamia ignava by biochemical tests and electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Facklamia tabacinasalis sp. nov., the type strain of which is CCUG 30090T.

Vagococcus lutrae sp. nov., isolated from the common otter (Lutra lutra).

Phenotypic and phylogenetic studies were performed on an unknown Gram-positive catalase-negative coccus isolated from a common otter (Lutra lutra). Comparative 16S rRNA gene sequencing demonstrated that the unknown bacterium represents a new subline within the genus Vagococcus, close to, but distinct from, Vagococcus fluvialis and Vagococcus salmoninarum. The unknown bacterium was readily distinguished from the two currently recognized Vagococcus species by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium be classified as a new species, Vagococcus lutrae, the type strain of which is CCUG 39187T.

Tessaracoccus bendigoensis gen. nov., sp. nov., a gram-positive coccus occurring in regular packages or tetrads, isolated from activated sludge biomass.

An isolate of a Gram-positive bacterium, designated strain Ben 106T, was obtained in pure culture by micromanipulation of a biomass sample obtained from a laboratory-scale sequencing batch reactor. This isolate grew axenically as cocci or clusters of cocci arranged in regular tetrads and was morphologically similar to the dominant organism observed in the biomass. This morphology resembled that of some Gram-positive and -negative bacteria and the so-called 'G-bacteria' commonly seen in activated sludge samples. Strain Ben 106T is a non-motile, facultative anaerobe. It is oxidase-negative, catalase-positive and is capable of reducing nitrate. This organism can grow between 20 and 37 degrees C, with an optimum temperature of 25 degrees C. The pH range for growth is between 6.0 and 9.0, with an optimum pH of 7.5. The isolate stained positively for intracellular polyphosphate granules. The diagnostic diamino acid of the peptidoglycan is LL-diaminopimelic acid (LL-A2pm) with a glycine moiety at position 1 of the peptide subunit, which characterizes the presence of a rare peptidoglycan (type A3-gamma'). Two menaquinones, MK-9(H4) and MK-7(H4), are present and the main cellular fatty acid is 12-methyltetradecanoic acid. The G + C content is 74 mol%. From phenotypic characteristics and 16S rDNA sequence analysis, the isolate differed sufficiently from its closest phylogenetic relatives, namely Propionibacterium propionicum, Propioniferax innocua, Friedmanniella antarctica, Luteococcus japonicus and Microlunatus phosphovorus in the A1 subdivision of the Gram-positive bacteria (i.e. Firmicutes with a high G + C content), suborder Propionibacterineae, to be placed in a new genus, Tessaracoccus, as Tessaracoccus bendigoensis gen. nov., sp. nov. The type strain is Ben 106T (= ACM 5119T).

Diversity of H2/CO2-utilizing acetogenic bacteria from feces of non-methane-producing humans.

The purpose of this work was to study H2/CO2-utilizing acetogenic population in the colons of non-methane-producing individuals harboring low numbers of methanogenic archaea. Among the 50 H2-consuming acetogenic strains isolated from four fecal samples and an in vitro semi-continuous culture enrichment, with H2/CO2 as sole energy source, 20 were chosen for further studies. All isolates were Gram-positive strict anaerobes. Different morphological types were identified, providing evidence of generic diversity. All acetogenic strains characterized used H2/CO2 to form acetate as the sole metabolite, following the stoichiometric equation of reductive acetogenesis. These bacteria were also able to use a variety of organic compounds for growth. The major end product of glucose fermentation was acetate, except for strains of cocci that mainly produced lactate. Yeast extract was not necessary, but was stimulatory for growth and acetogenesis from H2/CO2.

Ultrastructure of plaque associated with polytetrafluoroethylene (PTFE) membranes used for guided tissue regeneration.

The purpose of the study was to examine the ultrastructure of plaque contaminating polytetrafluoroethylene (PTFE) membranes used for guided periodontal tissue regeneration. 8 patients treated with Gore-Tex membranes received daily antibiotics (650 mg x 2 Femepen) and rinsed with 10 ml 0.2% chlorhexidine during a healing period of 30 days. Following retrieval, the membranes were processed for electron microscopy. External aspects of 12 portions from 4 partially exposed membranes were selected for detailed ultrastructural examination. The plaque-membrane interface was characterized by the presence of fibrin or discontinuous accumulation of intermicrobial matrix. Adjacent plaque-free areas of membrane surface exhibited no detectable electron-dense material. 3 structurally different groups of bacterial aggregations were observed on the strips: (i) dense layers of gram-positive cocci and rods dominated the external aspect of the open microstructure portion; (ii) cocci, rods and filamentous microorganisms embedded in fibrin filled the spaces of the open microstructure; (iii) a loosely arranged mixed microbiota consisting of gram-positive cocci and rods as well as of gram-negative microorganisms and spirochetes were present on the occlusive portion. Areas with morphologically intact bacteria alternated with areas with empty bacterial cell walls. One specimen also displayed degenerated Candida-like blastospores. This study shows that oral micro-organisms may colonize and extensively invade the open microstructure of PTFE material and that adhesion of plaque to the membrane surface is mediated either by fibrin or a discontinuous layer of intermicrobial matrix.