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Detection and quantification of microorganisms in a heterogeneous foodstuff by image analysis.

A relatively inexpensive image analysis system has been developed to semi-automate the detection and quantification of microbial growth in sections of food. A system based on an IBM PC compatible, with a frame store card, was programmed to scan Gram-stained sections using a motorized stage. Each field of view was thresholded after subtraction of a background image and the area between two thresholds measured. In the food studied it was found that, by using a size limit, it was possible to reduce the number of fields that needed to be examined by a microscopist to approximately 3% of those scanned. Visual examination was still required to distinguish bacterial cells from other stained objects which occasionally occur.

Algorithms↗

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi.

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Amino Acid Sequence↗

HIV-1 detection in cervicovaginal secretions during pregnancy.

OBJECTIVE: To assess the reproducibility of and factors associated with HIV detection in cervicovaginal secretions (CVS). DESIGN: Longitudinal study of 43 HIV-1-infected pregnant women in Paris. METHODS: HIV DNA was detected in peripheral blood mononuclear cells (PBMC) by Amplicor and gag nested polymerase chain reaction (PCR) assays. The HIV genotype was determined by heteroduplex mobility assay. Amplicor and gag nested PCR assays were performed on serial CVS samples for HIV DNA detection, and the HIV Monitor test was used for HIV RNA detection in plasma and CVS. RESULTS: A total of 144 CVS samples were collected from the women included in the study. HIV-1 DNA was detected in 36 (25%) of the 144 samples, from 16 (37.2%) of the 43 women. Results of HIV-1 DNA detection were concordant in the first two samples in 27 (84.4%) of the 32 women with at least two CVS samples. The last CVS sample collected in each woman was HIV-1 DNA-positive in 13 (30.2%) of the 43 women. Three factors were found to be independently associated with HIV-1 DNA detection in CVS: HIV-1 subtype B, absence of zidovudine therapy, and microbial cervicovaginal infection. HIV RNA was detected in CVS from 10 (23.3%) out of 43 women and correlated with DNA detection in the same sample and HIV RNA detection in plasma. CONCLUSIONS: DNA and RNA PCR can be used to detect HIV in cells and supernatants of CVS. These techniques may be useful in cohort studies to investigate HIV transmission and to evaluate the efficacy of antiretroviral drugs to reduce HIV excretion.

Adult↗

Emticicia oligotrophica gen. nov., sp. nov., a new member of the family 'Flexibacteraceae', phylum Bacteroidetes.

An aquatic bacterium, strain GPTSA100-15T, was isolated on nutritionally poor medium TSBA100 (tryptic soy broth diluted 100 times and solidified with 1.5 % agarose) and characterized using a polyphasic approach. The isolate was unable to grow on commonly used nutritionally rich media such as tryptic soy agar, nutrient agar and Luria-Bertani agar. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate was affiliated with the family 'Flexibacteraceae' in the phylum Bacteroidetes. Phylogenetically, it showed closest similarity (94.0 %) with an uncultured bacterial clone, HP1A92, detected in a sludge microbial community. Among the culturable bacteria, the isolate had highest 16S rRNA gene sequence similarity with Leadbetterella byssophila 4M15T (87.8 %). Sequence similarities with other members of the phylum Bacteroidetes were less than 85 %. The fatty acid profile of the isolate grown on TSBA100 indicated that the major fatty acid was iso-C15:0, which is also present in many members of the family 'Flexibacteraceae'. Cells of strain GPTSA100-15T are Gram-negative, strictly aerobic rods. The DNA G+C content of the isolate is 36.9 mol%. Results of phenotypic, chemotaxonomic and phylogenetic analyses clearly indicate that strain GPTSA100-15T represents a new genus within the family 'Flexibacteraceae'; the name Emticicia gen. nov. is proposed for the genus, with Emticicia oligotrophica sp. nov. as the type species. The type strain of Emticicia oligotrophica is GPTSA100-15T (=MTCC 6937T=DSM 17448T).

Bacterial Typing Techniques↗

Community proteomics of a natural microbial biofilm.

Using genomic and mass spectrometry–based proteomic methods, we evaluated gene expression, identified key activities, and examined partitioning of metabolic functions in a natural acid mine drainage (AMD) microbial biofilm community. We detected 2033 proteins from the five most abundant species in the biofilm, including 48% of the predicted proteins from the dominant biofilm organism, Leptospirillum group II. Proteins involved in protein refolding and response to oxidative stress appeared to be highly expressed, which suggests that damage to biomolecules is a key challenge for survival. We validated and estimated the relative abundance and cellular localization of 357 unique and 215 conserved novel proteins and determined that one abundant novel protein is a cytochrome central to iron oxidation and AMD formation.

Amino Acids↗

Microbial spoilage of whole sheep livers.

When liver treated with antibiotics to inhibit microbial growth were held at 10 degrees C, the initial high pH (6.4) declined as lactic acid accumulated throughout the storage period of 10 days. The glycogen content also declined, but the glucose concentration in the tissues remained high. When livers were allowed to spoil at 10 degrees C, distinct but variable floras developed within the tissues, in the drip, and on the upper surface. Deep-tissue floras were composed of anaerobic and facultative organisms (Lactobacillus, Enterobacter, Aeromonas); surface floras were generally dominated by strictly aerobic organisms (Pseudomonas, Acinetobacter); drip floras contained variable proportions of organisms of all three types, but the facultatively anaerobic Enterobacter were usually present as a major component. Spoilage occurred after 4 to 6 days with the development of visible discrete colonies on the upper surface without spoilage odors being evident. Changes in tissue and drip composition due to microbial activity could be detected only when spoilage had reached an advanced stage.

Animals↗

Use of a unique gene sequence as a probe to enumerate a strain of Bacteroides ruminicola introduced into the rumen.

Cloned fragments of genomic DNA from the ruminal anaerobe Bacteroides ruminicola subsp. brevis B14 were isolated and used as hybridization probes to identify closely related bacterial species. One DNA fragment unique to strain B14 was tested to determine its sensitivity in detecting homologous sequences among total ruminal microbial DNA. In a DNA titration experiment, the probe was capable of detecting strain B14 sequences in vitro down to 0.1% of the total bacterial DNA present in a hybridization assay. There was no detectable signal for total ruminal bacterial DNA. The specificity of this DNA fragment was exploited to enumerate strain B14 in a fresh mixed suspension of ruminal bacteria in vitro and after inoculation of the strain into the rumen. In vitro strain B14 had a half-life of 9 h. However, following inoculation into the rumen there was a very rapid loss of the strain to below the detectable limit within 3 h. The half-life was less than 30 min. This loss was not due to ruminal dilution or to bacteriophage attack but was possibly the result of a specific bacteriocinlike activity present in the rumen and detectable in fresh ruminal fluid.

Animals↗

Detection of viral pathogens by reverse transcriptase PCR and of microbial indicators by standard methods in the canals of the Florida Keys.

In order to assess the microbial water quality in canal waters throughout the Florida Keys, a survey was conducted to determine the concentration of microbial fecal indicators and the presence of human pathogenic microorganisms. A total of 19 sites, including 17 canal sites and 2 nearshore water sites, were assayed for total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, enterococci, coliphages, F-specific (F(+)) RNA coliphages, Giardia lamblia, Cryptosporidium parvum, and human enteric viruses (polioviruses, coxsackie A and B viruses, echoviruses, hepatitis A viruses, Norwalk viruses, and small round-structured viruses). Numbers of coliforms ranged from <1 to 1, 410, E. coli organisms from <1 to 130, Clostridium spp. from <1 to 520, and enterococci from <1 to 800 CFU/100 ml of sample. Two sites were positive for coliphages, but no F(+) phages were identified. The sites were ranked according to microbial water quality and compared to various water quality standards and guidelines. Seventy-nine percent of the sites were positive for the presence of enteroviruses by reverse transcriptase PCR (polioviruses, coxsackie A and B viruses, and echoviruses). Sixty-three percent of the sites were positive for the presence of hepatitis A viruses. Ten percent of the sites were positive for the presence of Norwalk viruses. Ninety-five percent of the sites were positive for at least one of the virus groups. These results indicate that the canals and nearshore waters throughout the Florida Keys are being impacted by human fecal material carrying human enteric viruses through current wastewater treatment strategies such as septic tanks. Exposure to canal waters through recreation and work may be contributing to human health risks.

Clostridium↗

Molecular machinations: chemokine signals in host-pathogen interactions.

Chemokines and their G-protein-coupled receptors represent an ancient and complex system of cellular communication participating in growth, development, homeostasis and immunity. Chemokine production has been detected in virtually every microbial infection examined; however, the precise role of chemokines is still far from clear. In most cases they appear to promote host resistance by mobilizing leukocytes and activating immune functions that kill, expel, or sequester pathogens. In other cases, the chemokine system has been pirated by pathogens, especially protozoa and viruses, which have exploited host chemokine receptors as modes of cellular invasion or developed chemokine mimics and binding proteins that act as antagonists or inappropriate agonists. Understanding microbial mechanisms of chemokine evasion will potentially lead to novel antimicrobial and anti-inflammatory therapeutic agents.

Animals↗

Transfer of infectious drug resistance in microbially defined mice.

Germ-free mice were intentionally associated with drug-resistant donor strains of Escherichia coli known to carry R factors and with drug-sensitive recipient strains. In vivo transfer of R factors was observed in all experiments, involving five different donor-recipient combinations. The number of converted recipients varied, depending upon the donor-recipient combination, but in all cases it was restricted by limiting numbers of either recipient or donor strains in the digestive tract of the microbially defined mice. Converted recipients were detected in fecal material as early as 5.5 hr after mice were associated with donor and recipient bacteria. Donors, recipients, and converted recipients were detectable in the stomach, small intestine, cecum, and large intestine of the microbially defined mice and their suckling young.

Animals↗

B-cell lymphomas differ in their responsiveness to CpG oligodeoxynucleotides.

Human B cells detect CpG motifs within microbial DNA via TLR9. Synthetic CpG oligodeoxynucleotides are currently being tested in clinical trials for the therapy of different types of B cell non-Hodgkin's lymphoma. However, there is only limited information on the CpG oligodeoxynucleotide sensitivity of primary malignant B cells of different non-Hodgkin's lymphoma entities. Here we found that most B-cell malignancies except plasmacytoma respond to CpG oligodeoxynucleotides by up-regulating expression of costimulatory and antigen-presenting molecules, by increasing expression of CD20, and by proliferation. In an in vitro analysis of 41 individual patient-derived primary tumor samples, B-cell chronic lymphocytic leukemia (B-CLL) and marginal zone lymphoma showed the strongest activation upon stimulation with CpG oligodeoxynucleotides. Small lymphocytic lymphoma, follicular lymphoma, mantle cell lymphoma, and large cell lymphoma showed an intermediate response. Consistent with CpG oligodeoxynucleotides sensitivity, TLR9 mRNA was present in B-CLL but absent in plasmacytoma. Although CpG oligodeoxynucleotides induced proliferation in all CpG oligodeoxynucleotide-sensitive types of B-cell malignancies, proliferation was weaker than in normal B cells and at least for B-CLL was followed by increased apoptosis. In conclusion, B-cell malignancies show significant differences in their responsiveness to CpG oligodeoxynucleotides. Focusing clinical studies on patients with highly CpG oligodeoxynucleotide-sensitive B-cell malignancies may improve the clinical outcome of such trials.

Adjuvants, Immunologic↗

Immunologic "memory" for microbial antigens in lymphocytes obtained from human bronchial mucosa.

Memory for previous immunologic contact with microbial antigens has been detected in lymphocytes from human bronchi as a secondary immune response, when tested in vitro. Antigens stimulated a predominantly proliferative response in blood lymphocytes that was significantly greater than the response in mucosal lymphocytes with purified protein derivative and Herpes simplex type 1 antigens. Co-culture experiments with autologous blood lymphocytes showed that cell-dependent suppression was one mechanism of the low response of bronchial lymphocytes. In the patient who inhaled a foreign body, a proliferative response to antigens was restricted to bronchus-associated lymphoid tissue lymphocytes, suggesting a recruitment of antigen-reactive cells from a circulating pool.

Antigens, Viral↗

Patient acceptance of Nordiject: a new drug delivery system for growth hormone.

Twenty-seven children, aged 5-20 years, with growth retardation were enrolled in an open study to evaluate the acceptability of a new injection pen (Nordiject 24, Novo Nordisk A/S) for administration of recombinant human growth hormone (r-hGH). Prior to this study, 17 of the patients had been receiving treatment with r-hGH administered via syringes and vials; their experiences with this conventional technique have been used for comparison with Nordiject. The injection pen is provided with a replaceable 27-gauge needle and a cartridge containing 24 IU of reconstituted r-hGH (12 IU/mL). A benzyl alcohol 0.9% solution is used as the solvent for the reconstitution. The pen can be regulated for doses from 0.5 to 8 IU per injection; regulation is accomplished by a turning movement. The patients had no problems with the dissolving procedure or handling the pen. Nearly two-thirds of the patients experienced less injection pain with the pen as compared with the syringe. Special attention was paid to local tolerability, which was reported to be good. The thigh was the preferred injection site. The residual contents from 68 cartridges returned by the patients were analyzed for microbial contamination. Contamination was detected in only one cartridge, which could not be confirmed in a repeated test. Because of the simplification of the injection procedure afforded by the injection pen, 20 of 27 patients were able to perform the injections themselves and hence assume a more active role in their treatment. The patients who previously had been using conventional syringes strongly preferred the pen, and all patients wished to continue using the device.

Adolescent↗

Hematological diseases-related mucormycosis: A retrospective single center study.

BACKGROUND AND AIM: Mucormycosis is a life-threatening invasive fungal infection. This study aimed to analyze the clinical characteristics of patients with hematologic malignancies complicated with mucormycosis. METHODS: This retrospective study investigated the clinical characteristics, epidemiological features, treatment, and prognosis of 46 patients with hematological diseases and Mucor infection as indicated by mNGS from August 28, 2020 to September 11, 2023. Metagenomic next-generation sequencing (mNGS) refers to the application of high-throughput sequencing technology for the comprehensive analysis of nucleic acid content in patient samples, facilitating the detection and characterization of microbial DNA and/or RNA, and then comparing and analyzing the results with an information database to determine the types of pathogenic microorganisms present in the sample. RESULTS: The median age of admission for the included patients was 49 years (9-78). Multivariate analysis identified age over 60 years (p&#x2009;=&#x2009;0.006&#x2009;<&#x2009;0.05), high-dose corticosteroids (p&#x2009;=&#x2009;0.001&#x2009;<&#x2009;0.05), neutropenia lasting more than 10 days (p&#x2009;=&#x2009;0.041&#x2009;<&#x2009;0.05), and two or more Mucor infections (p&#x2009;=&#x2009;0.004&#x2009;<&#x2009;0.05) were independent risk factors for OS in patients with hematological diseases. Moreover, differences between groups were analyzed using the Fisher exact probability method, and no significant difference was observed in the efficacy of various types of antifungal therapies. CONCLUSION: Patients with hematologic malignancies benefit greatly from early diagnosis and treatment when suspected of Mucor infection. mNGS is an important supplementary method for early diagnosis of Mucor infection. Moderated use of corticosteroids, reducing the duration of neutropenia, and enhancing autologous immune function are important measures to reduce patient mortality rate.

Retrospective Studies↗

Bronchoalveolar lavage fluid profiles in sarcoidosis, tuberculosis, and non-Hodgkin's and Hodgkin's disease. An evaluation of differences.

The aim of this study was to identify characteristic features in bronchoalveolar lavage fluid (BALF) samples of patients with tuberculosis, non-Hodgkin's or Hodgkin's disease and to investigate whether these differences facilitate the distinction of those disorders from sarcoidosis presenting with a similar clinical picture. Nonsmoker patients with histologically verified sarcoidosis (n = 29), tuberculosis (n = 6) proven by positive culture, non-Hodgkin's disease, (n = 6) or Hodgkin's disease (n = 7), both histologically verified, were investigated by BAL. A control group consisted of subjects without any pulmonary history. The presence of CD4+ and CD8+ T lymphocytes, as well as the CD4/CD8 ratio in BALF, aided in the differentiation between the various groups. Patients with malignant lymphomas had the lowest CD4/CD8 ratio in BALF, as well as in peripheral blood, and occasionally, plasma cells were present in BALF samples. The most important feature of BALF analysis in tuberculosis was detection of the causal microbial agent. In conclusion, although malignant lymphomas and tuberculosis require histologic evaluation and a positive culture, respectively, for diagnosis, BALF analysis may be of additional value in distinguishing those disorders from sarcoidosis.

Adolescent↗

Mutagenicity studies on urine concentrates from female users of dark hair color products.

Urines from women, collected before and after hair dyeing, were evaluated for mutagenic activity in the Ames Salmonella-Microsome Test. Thirty (30) sets of samples were tested as concentrates following removal of histidine on an XAD-2 resin column. None of the samples taken following the use of various Clairol products containing high levels of dyes gave any indication of increased mutagenic activity when compared to samples taken before hair dyeing. These results indicate that the use of hair dyes does not result in exposure of the urinary system to mutagens detectable in a sensitive microbial system, and thus suggest the absence of significant biological effects among users of hair dyes and persons occupationally exposed.

Female↗

Transmission of molecular information through electro-magnetic waves with different frequencies and its application to non-invasive diagnosis of patients as well as detection from patient's X-ray film of visible and not visible medical information: Part I.

Our previous study indicates the principle that information on the molecular structure and its quantity will be transmitted bi-directionally through a red-spectrum soft laser beam when specific molecules are placed in the close vicinity of the laser beam. The method was immediately applied for diagnosing diseases or localizing specific substances, using the Bi-Digital O-Ring Test, in moving or stationary animals or human subjects at clearly visible distances, without directly contacting the subject. This principle was also applied for the microscopic Bi-Digital O-Ring Test to examine cellular structures and substances within the cell at the magnified focused projected plane. The method was further expanded to an electron-microscopic Bi-Digital O-Ring Test, where, instead of a light beam as a source of electromagnetic wave carrier, an electron beam was used. Thus, it was possible to study the ultra-fine structure of cells. During the past several years, the author has been experimenting with the question of whether, instead of using visible light in the microscopic Bi-Digital O-Ring Test, if much shorter wavelengths, such as X-ray with strong penetrating force through living tissue, are used as the carriers of molecular information, and if X-ray pictures of the body are evaluated by a similar method as in the microscopic Bi-Digital O-Ring Test, molecular information existing in the pathways of the X-ray through the body might be detectable or not. Our studies indicate that, using X-ray film with good picture quality taken of specific parts of the body, one can detect not only specific microbial infections, such as bacterial, viral, or spirochete (e.g., Lyme), and changes in local chemistry including blood chemistry such as glucose, total cholesterol, uric acid, in major arteries or the heart, but also potentially effective medication. Using the Bi-Digital O-Ring Test resonance phenomenon between a reference control substance and an identical substance or its electromagnetic field imprint, anatomical structures of the soft tissue, such as blood vessels, nerves, and muscles can be identified even when they are not visible on the X-ray film because of the masking effect of other tissues with high density or large volume of tissue. Similar findings were also found in the CAT Scan and MRI pictures of normal and abnormal organs of the body. In this paper, two examples of such analyses, i.e. X-ray films of one patient with adenocarcinoma of the colon and another patient with rheumatoid arthritis of the knee joint are shown.

Arthritis, Infectious↗

The effect of nisin on the keeping quality of reduced heat-treated milks.

Milk was subjected to a combination process involving reduced heat treatment (RHT) of 117 degrees C for 2 s and nisin (75 and 150 IU ml(-1)). The microbial activity and other quality aspects were compared with a RHT control (without nisin) and with a ultrahigh temperature (UHT) milk processed at 142 degrees C for 2 s. Nisin was found to inhibit microbial growth for products stored without refrigeration, and RHT-nisin samples stored at 30 degrees C showed very low spoilage rates during 150 days, although not low enough to satisfy requirements for commercial sterility. RHT-nisin samples could be distinguished from and were preferred to the UHT control. Significant browning occurred during storage at 30 degrees C and above but was less in the RHT-nisin milk samples compared with the UHT milk. In RHT-nisin milk samples stored at 20 and 10 degrees C, no microbial activity could be detected in most samples after storage for 1 year. The effectiveness of this combination of RHT, nisin, and low storage temperatures against gram-positive spore-forming bacteria suggests potential for use of nisin in extended shelf life products.

Animals↗