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PubMed1985

Genotoxicity studies with mineral oils; effects of oils on the microbial mutagenicity of precursor mutagens and genotoxic metabolites.

In vitro genotoxicity assays are extensively used to predict carcinogenic activity in vivo. The standard microbial mutagenicity assays however often fail to yield positive results with mineral oils which are carcinogenic to mice in long-term skin-cancer studies. A comprehensive programme of studies has therefore investigated the basis of this apparently anomalous behaviour. This investigation has addressed the possible effects of oils on the bioactivation of precursor mutagens and the disposition of mutagenic metabolites by studying the microbial mutagenicity of selected precursor mutagens (benzo[a]pyrene, benzo[a]anthracene, 2-aminoanthracene and 2-naphthylamine) and intrinsically reactive mutagens [+/- )-benzo[a]pyrene-4,5-oxide and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) in the presence and absence of mineral oils. Notably the mutagenicity associated with the deliberate additions of these mutagens or precursor mutagens to oils was readily detected by the microbial assays. The mutagenicity of only one of the precursor mutagens, benzo[a]pyrene, was significantly reduced by the oils, and then only in the standard plate-incorporation assay. Interestingly the degree of suppression appeared to be related to the polycyclic aromatic hydrocarbon content of the oils. In the case of 2-aminoanthracene large enhancements in its mutagenicity were observed in the presence of oils. These latter findings appear to be due to effects of oils on the bioactivation of precursor mutagens rather than on the disposition of their bioactivation products. The mutagenicity of intrinsically reactive mutagens, of a type generated by bioactivation of polycyclic aromatic hydrocarbons, was not significantly reduced in the presence of mineral oils. This indicates that it is unlikely that components in oils trap or facilitate the deactivation of ultimate mutagens whether these pre-exist in the oil or are formed from precursors by bioactivation in the in vitro test system. Viewed overall these results suggest that mineral oils judged to be carcinogenic on the basis of in vivo studies in mouse skin may possess only very weak genotoxic potential. While this potential is likely to be a prerequisite for carcinogenic action, the current results cause attention to be focussed on other factors, e.g. promotion, as potentially important determinants of the carcinogenic potencies of mineral oils in mouse skin.

2-Naphthylamine↗

PubMed2003

Prospective study of the performance of vibrational spectroscopies for rapid identification of bacterial and fungal pathogens recovered from blood cultures.

Rapid identification of microbial pathogens reduces infection-related morbidity and mortality of hospitalized patients. Raman spectra and Fourier transform infrared (IR) spectra constitute highly specific spectroscopic fingerprints of microorganisms by which they can be identified. Little biomass is required, so that spectra of microcolonies can be obtained. A prospective clinical study was carried out in which the causative pathogens of bloodstream infections in hospitalized patients were identified. Reference libraries of Raman and IR spectra of bacterial and yeast pathogens highly prevalent in bloodstream infections were created. They were used to develop identification models based on linear discriminant analysis and artificial neural networks. These models were tested by carrying out vibrational spectroscopic identification in parallel with routine diagnostic phenotypic identification. Whereas routine identification has a typical turnaround time of 1 to 2 days, Raman and IR spectra of microcolonies were collected 6 to 8 h after microbial growth was detected by an automated blood culture system. One hundred fifteen samples were analyzed by Raman spectroscopy, of which 109 contained bacteria and 6 contained yeasts. One hundred twenty-one samples were analyzed by IR spectroscopy. Of these, 114 yielded bacteria and 7 were positive for yeasts. High identification accuracy was achieved in both the Raman (92.2%, 106 of 115) and IR (98.3%, 119 of 121) studies. Vibrational spectroscopic techniques enable simple, rapid, and accurate microbial identification. These advantages can be easily transferred to other applications in diagnostic microbiology, e.g., to accelerate identification of fastidious microorganisms.

Bacteria↗

PubMed1992

Effective cleaning and sanitizing of polysulfone ultrafiltration membrane systems.

Polysulfone UF membranes that were soiled by Cheddar cheese whey were successfully cleaned in place. This cleaning procedure was completed in about 1 h. Most cleaning chemicals used were common and inexpensive. The cleaning procedure consisted of rinsing the membrane system for 2 min with water initially and after each cleaning solution. Sodium hydroxide at pH 11.0, with .1% of a nonionic surfactant added, was circulated for 20 min. After a 2-min rinse with water, a 1:1 mixture of nitric and phosphoric acids at pH 2 was circulated for 20 min and rinsed again with water. Finally, sodium hydroxide at pH 11.0, with 200 ppm of sodium hypochlorite added, was circulated for 20 min and rinsed. All cleaning solutions and all rinse waters were at 54 degrees C. Membranes cleaned by this procedure were found to be free from whey residue under examination by scanning electron microscopy. The cleaning process did not damage the membranes even when it was used continuously for 300 h. Microbial populations on the membrane were estimated by incubating small (4-cm2) sections of membrane in screw-cap vials filled with trypticase soy broth. From the portion of vials showing growth after 72 h at 32 degrees C, a most probable microbial population was calculated. Santizing cleaned polysulfone UF membranes with 100 ppm of sodium hypochlorite or 100 ppm of dichloroisocyanurate at 54 degrees C resulted in membranes free from viable microorganisms. When dichloroisocyanurate was used at 10 degrees C and 200 ppm, a most probable microbial population of 290/m2 was found. No microbial growth was detected when cleaned and sanitized membranes were stored in tap water for 24 h. This technique for cleaning UF membranes does not require the use of a holding solution containing santizers to control the growth of residual microorganisms.

Bacteria↗

PubMed1998

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology.

Bacterial nonspecific acid phosphohydrolases (NSAPs) are secreted enzymes, produced as soluble periplasmic proteins or as membrane-bound lipoproteins, that are usually able to dephosphorylate a broad array of structurally unrelated substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Bacterial NSAPs are monomeric or oligomeric proteins containing polypeptide components with an M(r) of 25-30 kDa. On the basis of amino acid sequence relatedness, three different molecular families of NSAPs can be distinguished, indicated as molecular class A, B and C, respectively. Members of each class share some common biophysical and functional features, but may also exhibit functional differences. NSAPs have been detected in several microbial taxa, and enzymes of different classes can be produced by the same bacterial species. Structural and phyletic relationships exist among the various bacterial NSAPs and some other bacterial and eucaryotic phosphohydrolases. Current knowledge on bacterial NSAPs is reviewed, together with analytical tools that may be useful for their characterization. An overview is also presented concerning the use of bacterial NSAPs in biotechnology.

Acid Phosphatase↗

PubMed2002

Molecular studies of cerebrospinal fluid in human immunodeficiency virus type 1-associated opportunistic central nervous system diseases--an update.

Although the incidence of opportunistic central nervous system (CNS) diseases has markedly declined in developed countries following the advent of highly active antiretroviral therapies (HAARTs), they still represent a major diagnostic and therapeutic challenge over the world. The application of nucleic acid amplification techniques to the study of cerebrospinal fluid (CSF) has contributed substantially to their diagnosis. The detection of specific microbial genomes in the CSF is now the preferred test for some CNS opportunistic diseases, such as progressive multifocal leukoencephalopathy or cytomegalovirus encephalitis. More recent developments of these techniques are the quantitative amplification techniques and postamplification studies. Quantification of nucleic acids in CSF is an important aid both at the time of diagnosis, for the interpretation of positive findings, and during patient follow-up. Postamplification analyses can provide important information with regard to clinical patient management, e.g., detection of genotypic resistance to antimicrobial drugs, and in the attempt to elucidate disease epidemiology and pathogenesis.

AIDS Dementia Complex↗

PubMed1977

A radioimmunoassay for human antibody specific for microbial antigens.

A simple and sensitive method for detecting and quantitating antibody specific for microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and --log2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri, and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens.

Antibodies, Bacterial↗

PubMed1996

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure.

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.

Cell Wall↗

PubMed1996

Detection of viable Vibrio cholerae by reverse-transcriptase polymerase chain reaction (RT-PCR).

The use of conventional PCR can amplify target DNA from both viable and nonviable cells of Vibrio cholera. Detection of only viable microbial pathogens in biological samples, especially clinical and food samples, is usually desired to ensure positive test results are associated with active agents, and not the remains of dead cells. Positive identifications caused by nonliving causative agents may lead to misguided decisions concerning the effectiveness of treatment, and whether patient treatment should be continued or whether the food should be discarded. Consequently, this work was directed toward development of a reverse-transcriptase polymerase chain reaction (RT-PCR)-based in vitro DNA amplification method, which specifically detects only viable cells. Total RNA from both viable and nonviable cells was purified by using a FastPrep Cell Disrupter ([symbol: see text]Bio 101/Savant) and FastRNA extraction reagents ([symbol: see text]Bio 101). The purified RNA was treated with DNase I (RNase-free) to avoid any amplification from the contaminating target DNA. An RT-PCR approach using this rapid and effective method for RNA purification showed amplification of the target mRNA only from the viable cells. The sensitivity of detection of viable cells of V. cholerae was > or = 10(3), which is well within the minimum number of cells (10(5)-10(6)) required for infection. The use of a reliable prokaryotic RNA extraction method followed by RT-PCR amplification of the target mRNA can be used for specific detection of viable microbial pathogen, such as V. cholerae.

Base Sequence↗

PubMed2003

Toll-like receptors and their role in experimental models of microbial infection.

Effective host defense against microbial infection depends upon prompt recognition of pathogens, activation of immediate containment measures, and ultimately the generation of a specific and definitive adaptive immune response. The innate immune system of the host is responsible for providing constant surveillance against infection; when confronted by pathogens it deploys a series of rapidly acting antimicrobial effectors while simultaneously instructing the adaptive immune system as to the nature and context of the infectious threat. Pathogen recognition and activation of innate immunity is mediated by members of the Toll-like receptor (TLR) family through detection of conserved microbial structures that are absent from the host. Experimental models of infection using TLR-deficient mice, as well as limited human studies, have clearly demonstrated the critical role of TLRs in host defense against most major groups of mammalian pathogens.

Animals↗

PubMed2004

Changes of microbial flora and wound colonization in burned patients.

To determine time related changes of microbial colonization of burn wounds and body flora of burned patients, a prospective study was carried out. Fifty-one patients who were hospitalized at least 3 weeks were enrolled in the study. Periodic swabs were taken from burn wound, nasal, axillary, inguinal, and umbilical regions of the patients on admission and 7th, 14th, and 21st days of hospitalization. The mean body surface area burned was 22.9%. A total of 1098 microbial isolates were detected during the study period. Coagulase-negative staphylococci (CNS, 63.0%) and Staphylococcus aureus (19.7%) were the most prevalent isolates in admission cultures. There was a gradual decrease in the number of isolates of CNS and a marked increase in the numbers of S. aureus and Pseudomonas aeruginosa from admission to 21st day. At the 21st day, the most frequent organisms were S. aureus (37.6%), CNS (34.7%), and P. aeruginosa (16.2%). Methicillin resistance of staphylococci strains were increased constantly in study period. While 35.3% of burn wounds were sterile on admission, microbial colonization reached 86.3% within the first week. Nasal carriage of methicillin resistant S. aureus increased from 3.9% to 62.7% at 21st day. The nature of microbial wound colonization and flora changes should be taken into consideration in empirical antimicrobial therapy of burned patients.

Adolescent↗

PubMed2003

Potential for detection of microorganisms and heavy metals in potable water using electronic nose technology.

Studies have been carried out to determine the potential for the detection of different microbial species (Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa), alone and in the presence of low concentrations of different heavy metals (As, Cd, Pb and Zn) in bottled, reverse osmosis (RO) and tap water, using an electronic nose. Studies show that it is possible to discriminate control water samples from water contaminated with 0.5 ppm of a mixture of metals. The presence of heavy metals may modify the activity of microorganisms and thus the volatile production patterns. Bacterial species at 10(2)-10(4) colony forming units (CFUs) ml(-1) could be detected after 24 h of incubation. Work is in progress to identify the limits of detection for a range of other microorganisms, including, fungi and cyanobacteria, and chlorinated phenols using electronic nose technology.

Biomimetics↗

PubMed1998

Effects of sucralfate vs antacids on gastric pathogens: results of a double-blind clinical trial.

BACKGROUND: Unblinded studies suggested that sucralfate prophylaxis for stress ulcers is associated with a lower rate of nosocomial pneumonia than acid-reducing approaches. We performed a randomized, double-blind, double-sham clinical trial comparing the exact microbial effects of each treatment. METHODS: One hundred forty patients entered this study before major elective surgery, allowing baseline cultures of gastric and pulmonary secretions to be obtained intraoperatively. Postoperatively, the patients were treated with standard doses of either sucralfate or antacids, plus a sham of the other drug. Cultures were repeated twice daily for 3 days. Molecular epidemiological typing was used to track the appearance of specific microbes and their transmission from site to site, and clinical end points were compared. The number of patients chosen was for sufficient statistical power to detect differences in the microbial measures, as detecting differences in clinical measures would have required increasing the sample size by an order of magnitude. RESULTS: Gastric pH was affected by the form of stress ulcer prophylaxis throughout the study, and this pH effect affected the number of new gastric organisms appearing in the 2 different groups. Colonization of the airway with new gastric organisms occurred more frequently in the antacid than in the sucralfate group, and colonization of the airway with organisms of gastric origin was associated with occurrence of postoperative pneumonia. CONCLUSIONS: Both sucralfate and antacids offered safe and effective stress ulcer prophylaxis in this double-blind clinical trial of postoperative patients in an intensive care unit. In association with the drug's effects on gastric pH, more new pathogens appeared in the gastric contents of antacid-treated than sucralfate-treated patients.

Antacids↗

PubMed2006

Aerobic bacterial isolates from choledochal bile at a tertiary hospital.

We studied the aerobic bacterial isolates from bile and their susceptibilty pattern in patients with biliary tract disease. Samples of bile collected during endoscopic retrograde cholangio pancreatography (ERCP) and surgery, were inoculated on standard media. Bacterial identification and susceptibility were done by standard techniques. A total of 209 samples were cultured, out of which 128 samples showed growth. Total number of isolates obtained was 221. Poly-microbial infection was detected in 67 patients. Predominant aerobic bacterial isolates obtained were Escherichia coli 30% (67), Klebsiella species 23.98% (53), Enterococcus species 12.21% (27). Multi-drug resistance was noted in 57%. Higher resistance rate was noted among Gram negative bacilli for ampicillin (92.4%), cephalexin (82.46%), ciprofloxacin (68.42%), piperacillin (64.33%). Sensitivity to meropenem was 90.64% and amikacin was 76.61%. Gram positive bacteria showed high resistance to gentamicin (39.53%). Sensitivity to ampicillin was 86.05% and penicillin was 81.4%. Vancomycin and teicoplanin showed 100% sensitivity. From our study we conclude that E. coli, Klebsiella species and Enterococcus species are common pathogens infecting biliary tract. Poly-microbial infection and multi-drug resistance warrants culture and sensitivity to guide antimicrobial therapy. We recommend combination of amikacin and ampicillin for empirical therapy at our institution.

Adolescent↗

PubMed1989

Pharyngitis in adults: the presence and coexistence of viruses and bacterial organisms.

STUDY OBJECTIVE: To determine the presence and coexistence of viruses and bacterial organisms causing pharyngitis in adults. DESIGN: Open study using diagnostic methods, including rapid antigen-detection techniques, to test for the presence of viruses of the respiratory tract, as well as Mycoplasma pneumoniae. Chlamydia trachomatis, the Chlamydia species strain TWAR, and beta-hemolytic streptococci. SETTING: Open health care. PATIENTS: One hundred six consecutive adult patients, 15 to 65 years old, whose chief complaint was sore throat. MAIN RESULTS: Of the 106 patients, beta-hemolytic streptococci were found in only 24 patients (5 patients with group A streptococci, 13 with group C, 5 with group G, and 1 with group F); M. pneumoniae was found in 10 patients, the Chlamydia species strain TWAR in 9 patients, and viruses in 27 patients. Two microbes were simultaneously isolated in 3 patients, and no microbial findings were detected in 33 patients. CONCLUSION: Because 19 patients were infected with the Chlamydia species strain TWAR and M. pneumoniae, and 24 patients were infected with beta-hemolytic streptococci, the diagnostic procedures and therapies for adult patients with pharyngitis need to be reconsidered. The results of our study also confirm earlier suggestions that the Chlamydia species strain TWAR alone is a causative agent for pharyngitis in adults.

Adolescent↗

PubMed2004

Detection of Bacillus spores using PCR and FTA filters.

Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.

Bacillus↗

PubMed1985

Observations of barophilic microbial activity in samples of sediment and intercepted particulates from the demerara abyssal plain.

To better understand the ecological significance of pressure effects on bacteria in the abyssobenthic boundary layer, experimental suspensions of sediments and sinking particulates were prepared from samples collected in boxcore and bottom-moored sediment traps at two stations (depth, 4,470 and 4,850m) in the Demerara abyssal plain off the coast of Brazil. Replicate samples were incubated shipboard at 3 degrees C and at both atmospheric and deep-sea pressures (440 or 480 atm [4.46 x 10 or 4.86 x 10 kPa]) following the addition of [C]glutamic acid (<10 mug liter) or yeast extract (0.025%) and the antibiotic nalidixic acid (0.002%). In seven of the eight samples supplemented with isotope, a barophilic microbial response was detected, i.e., substrate incorporation and respiration were greater under in situ pressure than at 1 atm (101.3 kPa). In the remaining sample, prepared from a sediment trap warmed to 24 degrees C before recovery, pressure was observed to inhibit substrate utilization. Total bacterial counts by epifluorescence microscopy decreased with depth in each sediment core, as did utilization of glutamic acid. Significant percentages of the total bacterial populations in cold sediment trap samples (but not the prewarmed one or any boxcore sample) were abnormally enlarged and orange fluorescing after incubation with yeast extract and nalidixic acid under deep-sea conditions. Results indicated that in the deep sea, barophilic bacteria play a predominant role in the turnover of naturally low levels of glutamic acid, and the potential for intense microbial activity upon nutrient enrichment is more likely to occur in association with recently settled particulates, especially fecal pellets, than in buried sediments.

Journal Article↗

PubMed1999

The anti-microbial activity of maggot secretions: results of a preliminary study.

The ability of larval secretions to kill or prevent the growth of a range of potentially pathogenic bacteria was investigated in a preliminary laboratory study. Marked anti-microbial activity was detected against Streptococcus A and B and Staphylococcus aureus. Some activity was also detected against Pseudomonas sp. and a clinical isolate of a resistant strain of S. aureus (MRSA). No evidence of inhibition was recorded against Enterococcus or the Gram-negative bacteria Escherichia coli and Proteus. These findings are consistent with clinical observations made by us and others that larvae of the common greenbottle are able to combat clinical infections in a variety of wound types including those caused by antibiotic-resistant strains of bacteria.

Animals↗

PubMed1998

Changes in microbiological and physicochemical properties of bee pollen by application of gamma irradiation and ozone treatment.

The effects of gamma irradiation and ozone treatment on microbiological and physicochemical properties of bee pollen were investigated. Gamma irradiation at 7.5 kGy reduced the total microbial loads below detection levels (>10(2) CFU g(-1)), but after ozone treatment of up to 18 ppm for 8 h the total aerobic bacteria were found in concentrations of more than 10(3) CFU g(-1). Physicochemical properties such as amino acid and fatty acid composition, thiobarbituric acid value, mineral content, and pigment were not significantly changed by gamma irradiation, whereas ozone treatment caused significant changes in fatty acid composition, thiobarbituric acid value, and pigment by lipid oxidation and decoloration (P < 0.05).

Amino Acids↗