Known Numbers Of Bacteria Research Articles

Rapid identification of a subset of foodborne bacteria in live-cell assays.

The detection and identification of microbial pathogens in meat and fresh produce play an essential role in food safety for reducing foodborne illnesses every year. A new approach based on targeting a specific sequence of the 16S rRNA region for each bacterium is proposed and validated. The probe complex consists of a C60, a conjugated RNA detector which targets a specific 16S rRNA sequence, and a complementary fluorescent reporter. The RNA detectors were designed by integrating NIH nucleotideand Vienna RNA Webservice databases, and their specificities were validated by the RDP database. Probe complexes were synthesized for identifying E. coli K12, E. coli O157: H7, S. enterica, Y. enterocolitica, C. perfringens, and L. monocytogenes. First, under controlled conditions of known bacterial mixtures, the efficiency and crosstalk for identifying the foodborne bacteria were quantified to be above 94% and below 5%, respectively. Second, experiments were designed by inoculating meat products by known numbers of bacteria and measuring the limit of detection. In one experiment, 225g of autoclaved ground chicken was inoculated with 9 E. coli O157:H7, where 6.8 ± 1.2 bacteria with 95% confidence interval were recovered. Third, by positionally printing probe complexes in microwells, specific microorganisms were identified with only one fluorophore. The proposed protocol is a cell-based system, can identify live bacteria in 15min, requires no amplification, and has the potential to open new surveillance opportunities.Key points• The identification of foodborne bacteria is enabled in live-cell assays.• The limit of detection for 100g of fresh chicken breast inoculated with 4 bacteria is 2.7 ± 1.4 with 95% confidence interval.• The identification of five bacteria in a coded microwell chip is enabled with only one fluorophore.

Biological preparation of highly effective immunomagnetic beads for the separation, concentration, and detection of pathogenic bacteria in milk

We introduce a system for the efficient separation and concentration of pathogenic bacteria using biologically prepared immunomagnetic beads. Amylose magnetic beads (AMBs) were synthesized by an enzymatic reaction of amylosucrase from Deinococcus geothermalis (DGAS). The simple and rapid conjugation of AMBs and antibodies was achieved by the MBP-SPG fusion protein. MBP (maltose binding protein) binds to the surface of an AMB owing to its intrinsic affinity to the di-glucose in the AMB. SPG (streptococcal protein G) fused to the MBP has specific affinity to the Fc region of the antibody. Anti-Escherichia coli O157 antibodies were conjugated to the AMBs through a MBP-SPG linker without any physical and chemical treatments. The efficiency of separation and concentration of the target E. coli O157:H7 by the functionalized AMBs was revealed by plating counting, conventional polymerase chain reaction (PCR), and real-time RCR analysis. The immuno-AMBs effectively separated and concentrated the target bacteria from a commercial milk sample spiked with known number of bacteria, which was then analyzed by PCR to a detection limit of 10CFU/mL. On the other hand, no PCR product was produced when milk was introduced directly to a PCR reaction. These results show that MBP-SPG is an effective linker and the resulting immuno-AMBs are capable of separating and concentrating the target bacteria from a food matrix.

Quantification and qualification of bacteria trapped in chewed gum.

Chewing of gum contributes to the maintenance of oral health. Many oral diseases, including caries and periodontal disease, are caused by bacteria. However, it is unknown whether chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize that chewing of gum can trap bacteria and remove them from the oral cavity. To test this hypothesis, we developed two methods to quantify numbers of bacteria trapped in chewed gum. In the first method, known numbers of bacteria were finger-chewed into gum and chewed gums were molded to standard dimensions, sonicated and plated to determine numbers of colony-forming-units incorporated, yielding calibration curves of colony-forming-units retrieved versus finger-chewed in. In a second method, calibration curves were created by finger-chewing known numbers of bacteria into gum and subsequently dissolving the gum in a mixture of chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer was analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were requested to chew gum up to 10 min after which numbers of colony-forming-units and total numbers of bacteria trapped in chewed gum were determined using the above methods. The qPCR method, involving both dead and live bacteria yielded higher numbers of retrieved bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria were maximal during initial chewing after which a slow decrease over time up to 10 min was observed. Around 108 bacteria were detected per gum piece depending on the method and gum considered. The number of species trapped in chewed gum increased with chewing time. Trapped bacteria were clearly visualized in chewed gum using scanning-electron-microscopy. Summarizing, using novel methods to quantify and qualify oral bacteria trapped in chewed gum, the hypothesis is confirmed that chewing of gum can trap and remove bacteria from the oral cavity.

Point-of-Care System for Detection of Mycobacterium tuberculosis and Rifampin Resistance in Sputum Samples

ABSTRACT Early detection of Mycobacterium tuberculosis complex (MTBC) and markers conveying drug resistance can have a beneficial impact on preventive public health actions. We describe here a new molecular point-of-care (POC) system, the Genedrive, which is based on simple sample preparation combined with PCR to detect MTBC and simultaneously detect mutation markers in the rpoB gene directly from raw sputum sample. Hybridization probes were used to detect the presence of the key mutations in codons 516, 526, and 531 of the rpoB gene. The sensitivities for MTBC and rpoB detection from sputum samples were assessed using model samples spiked with known numbers of bacteria prepared from liquid cultures of M. tuberculosis . The overall sensitivities were 90.8% (95% confidence interval [CI], 81, 96.5) for MTBC detection and 72.3% (95% CI, 59.8, 82.7) for rpoB detection. For samples containing ≥1,000 CFU/ml, the sensitivities were 100% for MTBC and 85.7% for rpoB detection, while for samples containing ≤100 CFU/ml, the sensitivities were 86.4% and 65.9% for MTBC and rpoB detection, respectively. The specificity was shown to be 100% (95% CI, 83.2, 100) for MTBC and rpoB . The clinical sputum samples were processed using the same protocol and showed good concordance with the data generated from the model. Tuberculosis-infected subjects with smear samples assessed as scanty or negative were detectable by the Genedrive system. In these paucibacillary patients, the performance of the Genedrive system was comparable to that of the GeneXpert assay. The characteristics of the Genedrive platform make it particularly useful for detecting MTBC and rifampin resistance in low-resource settings and for reducing the burden of tuberculosis disease.

THE ANTIBACTERIAL ACTIVITY OF A COMPLEX OF IODINE AND A NON-IONIC SURFACE-ACTIVE AGENT.

Abstract The antibacterial activity of a complex of iodine and cetomacrogol against Escherichia coli and Staphylococcus aureus has been compared with a system prepared by diluting an iodine solution in ethanol and potassium iodide. Known numbers of bacteria were mixed with known concentrations of iodine and viable counts were made at intervals thereafter. The activity of both preparations was the same at equal iodine concentrations. Killing, when it occurred, was rapid, the relation between the iodine concentration and the dry weight of the bacterial suspension apparently being the controlling feature. The presence of serum reduced the activity, the reduction being greater for Staph. aureus than for E. coli. Temperatures from 20–37° had no effect whilst reducing the pH to 4, or below, caused a marked increase in the activity.

Potential for broad applications of flow cytometry and fluorescence techniques in microbiological and somatic cell analyses of milk

Monitoring the quality and safety of milk requires careful analysis of microbial and somatic cell loading. Our aim was to demonstrate proof of the principle that flow cytometry (FCM), coupled with fluorescence techniques for distinguishing between cell types, could potentially be employed in a wide variety of biological assays relevant to the dairy industry. To this end, we studied raw milk samples and ultraheat-treated milk, into which known numbers of bacteria or mouse cells were inoculated. For bacterial analyses, protein and lipids were removed, whereas only centrifugal lipid clearing was needed for somatic cell analyses. Cleared samples were stained with fluorescent dyes or with bacterial-specific fluorescent-labeled oligonucleotides and analyzed by FCM. A fluoresceinated peptide nucleic acid probe enabled efficient enumeration of bacteria in milk. Dual staining of samples with fluorescent dyes that indicate live (5-cyanol-2,3-ditolyl tetrazolium chloride, CTC or SYTO 9) or damaged cells (oxonol or propidium iodide, PI) enabled determination of viable bacteria in milk. Gram-positive and -negative bacteria were distinguished using hexidium iodide and SYTO 13 in dual staining of cleared milk samples. An FCM-based method gave a good correlation ( r=0.88) with total microscopic counts of somatic cells in raw milk. The FCM method also correlated strongly ( r=0.98) with the standard Fossomatic method for somatic cell detection. We conclude that FCM, coupled with fluorescence staining techniques, offers potentially diverse and rapid approaches to biological safety and quality testing in the dairy industry. Potential application of flow cytometers to a broad range of assays for milk biological quality should make this instrumentation more attractive and cost effective to the dairy industry and indeed the broader food industry.

Nested primers improve sensitivity in the detection of Helicobacter pylori by the polymerase chain reaction

To investigate potential routes of spread of infection by the polymerase chain reaction (PCR) it is important that the technique is effective in the types of specimen to be investigated. To establish the limits of detection of Helicobacter pylori by PCR in clinical material from the gastric mucosa, faeces, dental plaque and oral rinses, samples were seeded with known numbers of bacteria. DNA extraction was followed by amplification with primers from the urease C gene. Nested primers were used to amplify the PCR product which was detected using a digoxigenin-labelled probe. Faeces or plaque inhibited the single reaction 10(2)-10(6) fold. A second amplification using nested primers and probing increased the sensitivity to a level similar to that obtained with pure culture. This method is potentially useful with less likelihood of false negative results when trying to detect H. pylori by PCR in highly contaminated, clinical material.

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli.

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.

A Comparison of Seven Microbiological Sampling Procedures for Hard-Cooked Eggs

Seven microbiological sampling methods for hard-cooked eggs were evaluated and compared. Fresh eggs within 24 h of opposition were cooked at 98 C for 22 min in water and then cooled in 31 C water. Eggs were dipped for 10 min in a physiological saline solution (.85% saline) 10 C cooler than the eggs and containing known numbers of bacteria. Eggs were then stored for 4 days at 6 C and sampled using one of seven rinse or blend procedures on whole eggs, shell, shelled egg contents, or shelled egg contents plus shell. Highest recoveries were made from the peeled egg plus shell rinse, shell rinse, whole egg blend, and shell blend. Lowest recoveries were found with the whole egg rinse, the peeled egg rinse, and the peeled egg blend. These results indicate that significant numbers of organisms are associated with the inner and outer shell surfaces as well as the egg contents surface (albumen). Satisfactory recoveries can be made with the peeled egg plus shell rinse or the whole egg blend. Whole egg rinsing of hard-cooked eggs does not appear to be satisfactory.

Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry.

A commercially available flow cytometer (Cytofluorograf) was used for the immunofluorescence (IF) analysis of spores of Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, using fluorescein-labelled antispore conjugates. The cytometer was modified to allow analysis of known numbers of bacteria. In attempting to identify the region of the cytometer fluorescence histogram associated with the presence of stained spores, evidence was produced for signal components due to antibody bound to extracellular antigens. Under some reaction conditions these components were large enough partially or completely to obscure the fluorescence distribution imputed to the spores. The results support the hypothesis that the fluorescence histogram for a bacterial suspension can be modified by subtracting the histogram of the cell-free centrifugation supernatant to provide a fluorescence distribution more representative of the bacteria themselves. Spore and vegetative forms of B. anthracis could be differentiated in the flow IF assay by comparing the peak and area (integral) values of the photomultiplier output. The 90 degrees scatter histograms of the stained spores and their cell-free supernatants were so alike in shape that it was not possible to ascribe a unique peak to the spores themselves. Overall, these results confirm the considerable potential of flow cytometry for the rapid and quantitative IF assay of bacterial populations.

A New Model of Bone Infection Used to Evaluate the Efficacy of Antibiotic-impregnated Polymethylmethacrylate Cement

An animal model of bone infection was designed for evaluation of the benefits of adding antibiotics to polymethylmethacrylate (PMMA) cement. Femora of New Zealand albino rabbits were exposed at the knee; the medullary canals were entered and the contents aspirated. A known number of bacteria was added to each femur before it was filled with either normal Surgical Simplex P bone cement or Surgical Simplex P antibiotic bone cement. The presence or absence of bone infection was documented by quantitative bacteriologic, roentgenographic, and histologic techniques. Simplex antibiotic bone cement prevented infection, even in the presence of 10(7) bacteria. The effectiveness of the antibiotic bone cement was correlated with the rapid release of high levels of erythromycin (14.1 micrograms/g) and colistin (11.3 micrograms/g) specifically at the site of bacterial contamination.

Quantification of bacteria in operation wounds--an experimental investigation with swab sampling in pigs.

Quantitative recovery by swab cultures of Staphylococcus aureus or Escherichia coli from experimental wounds in pigs was studied for assessment of wound contamination in elective surgery. The recovery rate declined rapidly over the first hour. Ten minutes after seeding the wounds with known numbers of bacteria, the recovery was respectively 0.7% and 4.1% for S. aureus and E. coli. Recovery was not affected by the size of the wound. Streaking the swab onto the culture plate, or rinsing the swab in saline and plating out the rinsing fluid immediately after sampling resulted in a three to four times higher yield than by using Stuart's transport medium for later plating. The results provide an indication of the discrepancy between the number of micro-organisms recovered from wounds in elective surgery and the number of micro-organisms required to produce suppurative infection in experiments on animals and in man.

Production of experimental brain abscess in the rat

Experimental evaluation of brain abscess has been inhibited by the lack of a simple and reproducible model in small animals. A stereotaxic headholder and slow infusion of 1 microliter of saline, containing a known number of bacteria, were used to produce brain abscess consistently in the rat. The natural history of the brain abscess produced by this technique closely simulated that found in the human clinical situation.

A study of disposable surgical masks

A combined in vivo and in vitro testing method for surgical masks is presented. The mask to be tested was placed over the nose and mouth of a manikin which was accurately constructed according to human measurements. The manikin was placed in a special counting chamber and a known number of bacteria were blown through the mask by a nebulizer and an automatic respirator, closely simulating a human sneeze or cough. The chamber was then evacuated completely through an Andersen sampler so that all bacteria in the chamber which had not been retained by the mask could be collected and counted within this sampler. The efficiency of the different masks was expressed as the percentage of bacteria retained by the masks as compared to a control when the manikin did not wear a mask. The set-up closely resembled that of a surgeon bent over a patient in the operating room when he coughs or sneezes while wearing a mask. The relative efficiency of the different masks was found to be in this descending order: polypropylene fibers (Filtron), polyester-rayon fibers (Aseptex), glass fibers (Deseret), and paper.

Investigations into the basis of microbial ecology in soil, illustrated with reference to growth of soil diphtheroids and Azotobacter in a model system.

Soil is regarded as consisting of a number of microbial habitats or ecological niches. Each niche is made up of three factors: substrate, environment, and time. A model system was used to simulate a niche reasoned to occur in field soils and to be occupied by heterotrophic bacteria. The system comprised a particulate substrate (dried yeast) mixed in a porous environment (sand) maintained over periods of up to 1 year. Yeast and sand were placed in special culture tubes which were inoculated with known numbers of bacteria suspended in mineral salt solution. This was distributed evenly through the sand by a suction method. The fate of single and mixed populations of three species of soil bacteria was followed by plate counts and microscopical observations. The bacteria were Arthrobacter globiformis, a second soil diphtheroid, and Azotobacter chroococcum. Both diphtheroids grew and persisted well either in single or mixed culture. Populations of azotobacter grown singly initially maintained their numbers, but then declined progressively; they declined more rapidly in association with the second soil diphtheroid, and abruptly to zero with A. globiformis.

Evaluation of Methods for Estimating Numbers of Rhizobia on Alfalfa Seed1

AbstractComparative studies were made to determine the relative efficiency of different laboratory tests in detecting Rhizobium meliloti when alfalfa seeds were inoculated with known numbers of bacteria. The bottomless bottle (modified Leonard) and crock techniques, which utilize more than one seed per container, overestimated the number of rhizobia present on alfalfa seeds due to the transfer of rhizobia among seedlings in the test container. The transfer effect probably was due to root growth accompanied by multiplication and movement of rhizobia. Positive results (nodulated plants) in a container indicated that at least one seed carried one rhizobium. When rhizobia were present on the seed, a container with one inoculated seed gave results that were not significantly different from the results obtained when all seeds were inoculated. The agar tube technique tended to underestimate the number of rhizobia present on alfalfa seeds because the anaerobic conditions in the support medium inhibited nodule formation. The sand tube technique gave the most accurate information.

THE REMOVAL OF BACTERIA FROM GLASS SURFACES WITH CALCIUM ALGINATE, GAUZE AND ABSORBENT COTTON WOOL SWABS

SUMMARY: Calcium alginate wool swabs were no more efficient or reliable than gauze or absorbent cotton wool swabs when tested on drinking glasses infected with known numbers of bacteria. The number of bacteria left on the glass after swabbing was determined by a modified roll tube method.

LOCALIZATION OF BACTERIA IN TISSUES OF LOWERED RESISTANCE

The secondary infection of a so-called sterile abscess, by the intravenous injection of streptococci, is an outgrowth of work on postoperative wound infection in which we are engaged. We are attempting to arrive at a method by which the incidence of such infections can be reduced. As part of this work we induced infection with a known number of bacteria in a surgical wound in a dog, and then irrigated the wound with various antiseptic and germicidal solutions. We observed that an ordinary wound would heal, after contamination with a certain number of bacteria, without gross evidence of infection, such as oozing of pus, pus pockets, prolonged induration or sloughing of tissue. We also noted that certain wounds copiously irrigated with antiseptic solutions, would become infected even though the number of bacteria in each wound was less than that which a wound would usually dispose of without becoming grossly infected.

FORMULA FOR ESTIMATING NUMBER OF CUBIC CENTIMETERS OF STANDARDIZED BACTERIAL EMULSION TO MAKE BACTERIAL VACCINE OF KNOWN STRENGTH

As many bacteriologic workers are notoriously poor mathematicians, perhaps a formula for the estimation of the correct number of cubic centimeters of a standardized bacterial emulsion necessary to be added to a certain number of cubic centimeters of a saline or oil solution to make a vaccine of a known number of bacteria per cubic centimeter may be helpful. Example: Thirty cubic centimeters of a vaccine, each cubic centimeter of which shall contain 500,000,000 bacteria, is required to be made from an emulsion, each cubic centimeter of which contains 10,000,000,000 bacteria. By mathematical calculation it will be found that this vaccine must be made up to 28.5 c.c. of saline solution + 1.5 cc. of the 10,000,000,000 standardized bacterial emulsion. As the resulting vaccine must consist of 30 c.c., then this 1.5 c.c. must be subtracted from the 30 c.c., giving 28.5 cc. of saline, to which the 1.5 are added.