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Detection of peptidoglycan and beta-glucan with silkworm larvae plasma test.

A method to detect peptidoglycan and (1-->3) beta-D-glucan with silkworm larvae plasma (SLP) derived from the hemolymph of the silkworm, Bombyx mori was developed. SLP contains all of the factors of the pro-phenol oxidase cascade, an important self-defense mechanism of insects. Peptidoglycan or (1-->3)-beta-D-glucan initiates the cascade, in which pro-phenol oxidase is finally activated to phenol oxidase. The phenol oxidase activity was colorimetrically or visually detected with 3,4-dihydroxyphenylalanine as a substrate. SLP displayed high reactivity with peptidoglycan and polysaccharides containing 1,3-beta-glucosidic linkages, but not with endotoxins. SLP is useful for the detection of microbial contamination because peptidoglycan and (1-->3)-beta-D-glucan are cell wall components of bacteria and fungi, respectively.

Animals↗

Validation of AKACID plus as a room disinfectant in the hospital setting.

AKACID Plus, a novel polymeric guanidine with broad antimicrobial activity against multiantibiotic-resistant bacterial strains, was used in the present study as a room disinfectant. Disinfection of closed rooms experimentally contaminated with antibiotic-susceptible and multiresistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Escherichia coli was performed using AKACID Plus at concentrations of 0.1, 0.25, and 0.5% for 100 min. Bacterial suspensions were distributed on plastic and stainless steel plates and placed in a test room. Recovery of the test microorganisms was determined before nebulizing, 60 and 100 min after initiation, and 4 h after the end of room disinfection by a simple swab-rinse technique. The swab-rinse method demonstrated a dose- and time-dependent effectiveness of AKACID Plus in eradicating S. aureus, E. coli, and P. aeruginosa on plastic and stainless steel plates. Nebulized 0.5% AKACID Plus was successful in eliminating all hospital pathogens within 340 min. After the use of 0.25% AKACID Plus, MRSA was still detectable on microbial carrier plates. The test concentration of 0.1% AKACID Plus achieved a significant reduction of S. aureus and P. aeruginosa on plastic and stainless steel plates but was sufficient to eradicate only E. coli. These results suggest that nebulized AKACID Plus at a concentration of 0.5% is a potent substance for eradication of pathogenic organisms in the hospital setting.

Disinfectants↗

Bioluminescence for USP sterility testing of pharmaceutical suspension products.

Bioluminescence measurement significantly improved the accuracy, sensitivity, precision, and reliability of the current visual endpoint determination for the USP sterility test and eliminated the day 7 transfer/dilution step required for testing suspension products. Thirteen strains of bacteria and fungi (representing potential contaminants in sterile products), three pharmaceutical suspension products, and four media were used in the experiment. No interference from suspension products was encountered in the detection of microbial growth by the bioluminescence measurement. The poor fungal growth encountered was attributed to insufficient diffusion of oxygen into the medium and was circumvented by use of a large tube size (38 by 200 mm) or by vortexing the medium once during the 2-week incubation period. Bioluminescence measurement would facilitate automated handling of the sterility test endpoint readout operation. The optimum parameters of bioluminescence measurement for application in sterility testing were determined.

Bacteria↗

Spatial changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores.

Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Microsensor measurements showed that oxygen was depleted from 140 microM at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-microm-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique. The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth. 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone. Comparative sequence analysis of these clones identified members of the alpha and beta subclasses of Proteobacteria as the abundant populations in the oxic zone. In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil. The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis. This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined.

Bacteria↗

Evidence for the presence of Simkania negevensis in drinking water and in reclaimed wastewater in Israel.

Simkania negevensis is a recently discovered chlamydia-like intracellular microorganism which has been associated with bronchiolitis in infants and with community-acquired pneumonia in adults; a high seroprevalence of antibodies to the microorganism has been found in various population groups. S. negevensis can be grown in various cell lines as well as in free-living amoebae such as Acanthamoeba polyphaga. In this study, evidence for the existence of Simkania or Simkania-like microorganisms in drinking water and in reclaimed wastewater is presented for the first time. Detection of the microorganism was made possible by the development of a specific and sensitive filter membrane immunoassay and was confirmed by PCR detection of microbial DNA in the water samples. The common presence of S. negevensis in water sources together with the high seroprevalence of antibodies to it and early age of acquisition of infection may implicate water as a source of infection. The possible significance of this finding for public health and for municipal water testing and treatment needs to be further examined.

Acanthamoeba↗

Use of Peptide library screening to detect a previously unknown linear diagnostic epitope: proof of principle by use of lyme disease sera.

Diagnostic peptides previously isolated from phage-displayed libraries by affinity selection with serum antibodies from patients with Lyme disease were found to give reproducible serum reactivity patterns when tested in two different enzyme-linked immunosorbent assay formats. In addition, the hypothetical possibility that peptides selected by this type of "epitope discovery" technique might identify the original antigens eliciting antibody responses was tested by searching for sequence similarities in bacterial protein databases. In support of this hypothesis, our search uncovered similarities between peptides representing two different sequence motifs and sequences in the VlsE and BBA61 antigens of Borrelia burgdorferi. Utilizing synthetic peptides, we verified that the sequence KAASKETPPALNK, located at the C terminus of the VlsE antigen, had the same reactivity pattern to sera from patients with extracutaneous Lyme disease as the diagnostic peptide SKEKPPSLNWPA, with which it shared a 7-amino-acid-residue match (consensus residues are underlined). A peptide with conservative mutations of five of the consensus residues was nonreactive, strongly suggesting that the VlsE sequence represents the epitope that originally elicited antibody responses in these patients. The diagnostic sensitivity of this new VlsE epitope was relatively low (30%) compared to that (100%) of the well-documented C6 diagnostic peptide of VlsE when tested in our small cohort of 10 patients with Lyme disease. Nonetheless, the identification of this previously unknown epitope serves as a proof of the principle of the hypothetical ability of "epitope discovery" techniques to detect specific microbial antigens with diagnostic relevance in infectious diseases.

Amino Acid Sequence↗

Clinical comparison of the recoveries of bloodstream pathogens in Septi-Chek brain heart infusion broth with saponin, Septi-Chek tryptic soy broth, and the isolator lysis-centrifugation system.

The recoveries of microorganisms in the Septi-Chek biphasic tryptic soy broth (TSB) and Isolator blood culturing systems were compared with the recovery in Septi-Chek biphasic brain heart infusion broth supplemented with the lytic agent saponin (L-BHIB; Roche Diagnostics, Division of Hoffmann-La Roche Inc.). A total of 5,096 TSB-L-BHIB blood culture sets and 1,778 Isolator-L-BHIB sets were evaluated. There were 435 pathogenic organisms recovered in 413 (8.1%) of the TSB-L-BHIB cultures, with 348 organisms in the TSB bottles and 370 in the L-BHIB bottles (P less than 0.05). Significantly more yeast isolates were recovered in the L-BHIB system (P less than 0.01). Terminal subculture of the L-BHIB system was required to detect 13 clinically significant organisms. Significantly more organisms were recovered in the L-BHIB bottles than with the Isolator system (P less than 0.001), including Torulopsis glabrata, coagulase-negative staphylococci, and Pseudomonas species. The time required for detection of all major groups of organisms was the same in both comparisons. However, detection of microbial growth in the L-BHIB bottles required macroscopic inspection of the attached agar slide unit and the use of terminal subcultures after 7 days of incubation. The primary advantage of the biphasic L-BHIB system when compared with either the biphasic TSB system or the Isolator system was improved recovery of fungi.

Bacteria↗

Comparison of BACTEC Plus 26 and 27 media with and without fastidious organism supplement with conventional methods for culture of sterile body fluids.

We compared the BACTEC Plus 26/27 culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) with and without fastidious organism supplement with conventional centrifugation preparation and plating for the recovery and speed of detection of microorganisms. A total of 1,101 sterile body fluid specimens were collected and processed at five hospital laboratories, yielding 234 (21%) positive cultures. Of the 176 isolates considered clinically significant, 133 (76%) were recovered by both the BACTEC system and conventional culture, while 28 (16% [P < 0.005]) were recovered by BACTEC only and 11 (6%) were recovered by conventional culture alone. There were no statistically significant differences in the speed of detection of microbial growth. It was found that BACTEC, with or without the addition of fastidious organism supplement, exhibited improved sensitivity for the recovery of microorganisms, including fastidious bacteria.

Bacteria↗

Infrared color photomicrography of soil microorganisms.

The infrared color photomicrography technique for detecting unstained microbial cells in soil is revised for use with Kodak's recently introduced Ektachrome infrared film. Thes new version of the film required achromatic objectives instead of apochromats, and the use of a blue filter in addition to the previously required red filter. These filters also improve the visual focus capability of the operator. It was found that dry soil smears can be photographed as either immersion oil or aqueous mounts. Several makes of bright-field transmitted-light microscopes gave similar results. Phase microscopy, however, produced deteriorated image quality although the proper 'false' colors were produced.

Bacteria↗

The use of gas-sensor arrays to diagnose urinary tract infections.

Sensorial analysis based on the utilisation of human senses, is one of the most important and straightforward investigation methods in food and chemical analysis. An electronic nose has been used to detect in vivo Urinary Tract Infections from 45 suspected cases that were sent for analysis in a UK Health Laboratory environment. These samples were analysed by incubation in a volatile generation test tube system for 4-5 h. The volatile production patterns were then analysed using an electronic nose system with 14 conducting polymer sensors. An intelligent model consisting of an odour generation mechanism, rapid volatile delivery and recovery system, and a classifier system based on learning techniques has been considered. The implementation of an Extended Normalised Radial Basis Function network with advanced features for determining its size and parameters and the concept of fusion of multiple classifiers dedicated to specific feature parameters has been also adopted in this study. The proposed scheme achieved a very high classification rate of the testing dataset, demonstrating in this way the efficiency of the proposed scheme compared with other approaches. This study has shown the potential for early detection of microbial contaminants in urine samples using electronic nose technology.

Algorithms↗

Microbial contamination of antiseptic-soaked cotton balls.

We investigated microbial contamination of in-use antiseptics at a hospital. No microbial contamination was observed in 70 samples of 0.02% benzalkonium chloride solution (500-ml volume), 70 samples of 1% titratable I2 povidone-iodine solution (250-ml volume), or 15 samples of 0.1% ethacridine lactate solution (500-ml volume) during use in reduced amounts. Nor was any microbial contamination observed in 70 samples of cotton balls soaked in 1% titratable I2 povidone-iodine solution in canisters or cotton gauze soaked in 70% (w/v) ethanol solution in canisters. However, among 70 samples of cotton balls soaked in 0.02% benzalkonium chloride solution in canisters, 6 (8.6%) were contaminated with 10(4) to 10(6) viable cells/ml. The microbial species detected were glucose non-fermentative bacilli such as Alcaligenes xylosoxidans and Pseudomonas putida. The contaminants obtained from cotton balls soaked in 0.02% benzalkonium chloride solution did not proliferate in that solution or in distilled water but showed rapid growth in the cotton balls soaked in either of these liquids. These findings suggested that benzalkonium chloride solution tends to become contaminated when cotton balls are immersed. Therefore, cotton balls soaked in benzalkonium chloride solution are not recommended as an antiseptic. When no other choice is available, the cotton balls should be soaked in benzalkonium chloride solution at the time of usage.

Alcaligenes↗

Rapid separation of microorganisms by quartz microchip capillary electrophoresis.

We developed and optimized a system coupling microchip capillary electrophoresis (MCE) and laser-induced fluorescence (LIF) detection for the analysis of microorganisms. The MCE-LIF system successfully separated pure cultures of lactic acid bacteria and Saccharomyces cerevisiae within 200 s. The results indicate that the MCE system can be conveniently used for the rapid and highly sensitive detection of microorganisms. Thus, MCE can provide a cheap and simple method for the on-line detection of microbial contamination.

Bacteria↗

Signalment and clinical features of diskospondylitis in dogs: 513 cases (1980-2001).

OBJECTIVE: To determine signalment, clinical features of the disease, and treatment in dogs with diskospondylitis. DESIGN: Case-control study. ANIMALS: 513 dogs with diskospondylitis (cases) and 236,109 canine hospital accessions (controls) from 12 veterinary teaching hospitals. PROCEDURE: Information retrieved from the medical records of 123 dogs with diskospondylitis at the Louisiana State University veterinary teaching hospital between 1980 and 2001 included sex, age, breed, primary complaint, neurologic status, location of lesions, causative organism, treatment, and outcome. The signalment of 390 additional cases from 11 other veterinary teaching hospitals was accessed from the Veterinary Medical Database. Comparisons were made with controls from the same time periods. RESULTS: Male dogs were twice as likely as female dogs to be affected (odds ratio [OR], 2.0; 95% confidence interval [CI], 1.7 to 2.4). Dogs were significantly more likely to be affected as age increased. Purebred dogs, especially Great Danes, were more likely than mixed-breed dogs to be affected (OR, 73; CI, 4.3 to 12.6). For dogs from Louisiana State University, Staphylococcus spp, Brucella spp, Streptococcus spp, and Escherichia coli were isolated most often; multiple organisms were detected via microbial culture in 11 dogs. The mean duration of treatment was 53.7 weeks. CONCLUSIONS AND CLINICAL RELEVANCE: Male dogs, older dogs, and Great Danes appeared more likely to be affected with diskospondylitis than female dogs, dogs < 1 year of age, and mixed-breed dogs, respectively. Long-term administration of antimicrobial drugs for treatment of diskospondylitis may be expected. Identification of the causative organism and early treatment are recommended.

Age Factors↗

A subset of Toll-like receptor ligands induces cross-presentation by bone marrow-derived dendritic cells.

Dendritic cells (DCs) are capable of cross-presenting exogenous Ag to CD8(+) CTLs. Detection of microbial products by Toll-like receptors (TLRs) leads to activation of DCs and subsequent orchestration of an adaptive immune response. We hypothesized that microbial TLR ligands could activate DCs to cross-present Ag to CTLs. Using DCs and CTLs in an in vitro cross-presentation system, we show that a subset of microbial TLR ligands, namely ligands of TLR3 (poly(inosinic-cytidylic) acid) and TLR9 (immunostimulatory CpG DNA), induces cross-presentation. In contrast to presentation of Ag to CD4(+) T cells by immature DCs, TLR-induced cross-presentation is mediated by mature DCs, is independent of endosomal acidification, and relies on cytosolic Ag processing machinery.

Adaptor Proteins, Signal Transducing↗

[Bacteriological features of peritonitis in perforative gastroduodenal ulcer].

Bacteriological investigation of 137 probes of the exudate from the abdominal cavity has revealed positive results in 60.6%. The detection of microbial flora was the more frequent the greater was the spread and degree of peritonitis. The monoculture was revealed in 92.8%, the colibacillus being the most frequent. The isolated microbial flora was sensitive to antibiotics of the cephalosporin group.

Bacteria↗

[Molecular diagnosis of infections].

Molecular diagnostics (detection of nucleic acids by molecular genetics techniques) become more valuable in clinical diagnosis of disease. Apart from the already long-time used genetic techniques for detection of congenital anomalies, current use of molecular techniques includes detection of microbial pathogens. The character of these techniques increases the possibility of achieving diagnosis in cases where classical cultivation is not possible, is not reliable or is not fast enough. As with every new approach, molecular diagnostics have faced encountered reactions from the scientific community. Some scientists tend to overestimate the value of molecular diagnostic techniques, while sceptics, sometimes influenced by a biased or incomplete knowledge of the technology, think it is of little value. In this work, on the basis of literature and our own data from more than 5 years of experience with these methods, we have assessed the pros and cons of the use of molecular diagnostics of infectious diseases in the light of their potential use in clinical practice.

Bacterial Infections↗

[Antiinfective host defense mechanism: toll-like receptors and innate immunity].

The innate immune system has evolved as the first line of defense against invading microorganisms. The recent discovery of the toll-like receptors(TLRs) has rapidly expanded our knowledge of molecular events that initiate host-pathogen interactions. The TLRs, which are expressed on the surface of cells, involved in innate immune recognition, including macrophages and dendritic cells, have a crucial role in the detection of microbial infection. Signals initiated by the interaction of TLRs with pathogen-associated molecular patterns (PAMPs) induce activation of the inflammatory and antimicrobial innate immune response. Ten members of the TLR family have been identified, and they appear to recognize PAMPs, including lipopolysaccharide, peptidoglycan, and bacterial DNA. There has been considerable interest in how adaptive immune responses are controlled by the innate immune system. Recent studies have suggested that TLRs may control the induction of Th1 responses and that a separate system of recognition regulates the Th2 response. Thus TLR signaling represents a key component in the innate immune response to microbial infection.

Animals↗

[Development and significance of occupational and iatrogenic contact sensitization in nummular microbial eczema].

The pathogenesis of the epidermal and cutaneous reactions to a microbic invasion comprises many criteria for the diagnosis of a nummular microbial eczema. Detection of the focus, healing of the eczema after focal sanitation, positive reactions to bacterial antigens, decrease of Igm and IgG, and other parameters represent the cutaneous component of the disease, while the lack of epidermal sensitizations to low-molecular haptenes and the predominantly negative epicutaneous tests constitue the accentuating criteria. The results obtained in 95 patients with nummular microbic eczema show the part played by epidermal alterations in the development of occupational an iatrogenic contact sensitizations. Moreover, these alterations allow to recognize to what extent these dermatoses are due to occupational causes.

Antigens, Bacterial↗