Enumeration Tests Research Articles

Prevalence and Distribution of Campylobacter jejuni in Small-Scale Broiler Operations

Campylobacter jejuni has been recognized as one of the most prevalent causes of foodborne bacterial illnesses in humans. Previous studies have focused on the transmission routes of C. jejuni from commercial flock farms to the final retail product. The objective of this study was to determine the prevalence of C. jejuni and Campylobacter spp. in eggshells, live birds, feed, drinking water, and the rearing environment in a small-scale broiler operation. Broilers were raised under two different production systems: (i) environmentally controlled housing and (ii) open-air housing with two replications. Each week, samples were collected from eggshells, bird feces, feed, drinking water, enclosures (vertical walls of bird housing), and feed troughs for enumeration and isolation testing. All samples were plated on modified charcoal-cefoperazone-deoxycholate agar to determine the log CFU per gram and percent prevalence of Campylobacter spp. Isolation of C. jejuni was verified with latex agglutination and hippurate hydrolysis tests. The results from this study suggest that vertical transmission of these bacteria from egg surfaces to newly hatched chicks is not a significant risk factor. The results also suggest that the prevalence of C. jejuni at time of harvest (week 6) was significantly higher (P < 0.05) in the open-air housing broilers than in those in the environmentally controlled housing. Elevated levels of cross-contaminants, especially water and feed, may have played a role in this outcome.

Surface chemical and biological characterization of flax fabrics modified with silver nanoparticles for biomedical applications.

Silver nanophases are increasingly used as effective antibacterial agent for biomedical applications and wound healing. This work aims to investigate the surface chemical composition and biological properties of silver nanoparticle-modified flax substrates. Silver coatings were deposited on textiles through the in situ photo-reduction of a silver solution, by means of a large-scale apparatus. The silver-coated materials were characterized through X-ray Photoelectron Spectroscopy (XPS), to assess the surface elemental composition of the coatings, and the chemical speciation of both the substrate and the antibacterial nanophases. A detailed investigation of XPS high resolution regions outlined that silver is mainly present on nanophases' surface as Ag2O. Scanning electron microscopy and energy dispersive X-ray spectroscopy were also carried out, in order to visualize the distribution of silver particles on the fibers. The materials were also characterized from a biological point of view in terms of antibacterial capability and cytotoxicity. Agar diffusion tests and bacterial enumeration tests were performed on Gram positive and Gram negative bacteria, namely Staphylococcus aureus and Escherichia coli. In vitro cytotoxicity tests were performed through the extract method on murine fibroblasts in order to verify if the presence of the silver coating affected the cellular viability and proliferation. Durability of the coating was also assessed, thus confirming the successful scaling up of the process, which will be therefore available for large-scale production.

Comparison of the new Mycofast Revolution assay with a molecular assay for the detection of genital mycoplasmas from clinical specimens

BackgroundGenital mycoplasmas are opportunistic bacteria that are associated with undesirable gynaecologic and reproductive events. Mycoplasmas are fastidious bacteria with increasing resistance to routine antimicrobials and often fail to grow on conventional culture methods. The commercial Mycofast Revolution assay permits the phenotypic detection and identification of genital mycoplasmas. Antimicrobial susceptibility testing against five antimicrobial agents with MICs corresponding to the CLSI guidelines can also be performed. This study aimed to compare the new commercially available Mycofast Revolution assay with a multiplex PCR assay.MethodsSelf-collected swabs were obtained from pregnant women attending the antenatal clinic of a tertiary academic hospital in Pretoria, South Africa from October 2012 to November 2012. These swabs were used to seed UMMt and modified Amies transport media. The seeded UMMt transported medium was used to inoculate the Mycofast Revolution assay for the identification, enumeration and antimicrobial susceptibility testing of genital mycoplasmas. Following DNA extraction from the modified Amies transport medium, specimens were subjected to a multiplex PCR assay for the detection of genital mycoplasmas.ResultsThe Mycofast Revolution kit had a sensitivity and specificity of 77.3% (95% CI: 62.15% to 88.51%) and 80% (95% CI: 28.81% to 96.70%), respectively, against the PCR assay. The positive and negative predictive values were 97.1% (95% CI: 85.03% to 99.52%) and 28.6% (95% CI: 8.57% to 58.08%). Genital mycoplasmas were detected in 71.4% (35/49) of samples with the Mycofast Revolution assay with 49% (24/49) being Ureaplasma spp. and 22.4% (11/49) mixed strains. The multiplex PCR assay had a positivity rate of 89.8% (44/49) for genital mycoplasmas; mixed strains were present in 51% (25/49) of samples, Ureaplasma spp. in 16.3% (8/49) and M. hominis in 22.4% (11/49) of samples.ConclusionsThere was a fair agreement (κ = 0.319) between the Mycofast Revolution assay and the mPCR assay. With the high prevalence rates of genital mycoplasmas, fast and efficient diagnostic methods are imperative to treat infections and minimise complications. The Mycofast Revolution assay is simple to use, has a short turn-around time and interpretation of results are straightforward. This assay circumvents common problems experienced with conventional culture and molecular methods in diagnostic laboratories where skilled personnel are limited and can be used as an alternative diagnostic assay.

A comparison of test statistics for the recovery of rapid growth‐based enumeration tests

This paper considers five test statistics for comparing the recovery of a rapid growth-based enumeration test with respect to the compendial microbiological method using a specific nonserial dilution experiment. The finite sample distributions of these test statistics are unknown, because they are functions of correlated count data. A simulation study is conducted to investigate the type I and type II error rates. For a balanced experimental design, the likelihood ratio test and the main effects analysis of variance (ANOVA) test for microbiological methods demonstrated nominal values for the type I error rate and provided the highest power compared with a test on weighted averages and two other ANOVA tests. The likelihood ratio test is preferred because it can also be used for unbalanced designs. It is demonstrated that an increase in power can only be achieved by an increase in the spiked number of organisms used in the experiment. The power is surprisingly not affected by the number of dilutions or the number of test samples. A real case study is provided to illustrate the theory.

FMCS: a novel algorithm for the multiple MCS problem

Clustering and classification of large-scale chemical data are essential for navigation, analysis and knowledge discovery in a wide variety of chemical application domains. The maximum common structure (MCS) for a group of compounds is an important element of such classification, providing insight into activity patterns and enabling scaffold alignment for a more consistent 2D depiction. Most modern, exact MCS implementations use back-tracking [1] or clique detection [2], and handle the multiple MCS problem by recursive reduction to successive pairwise maximal common substructure searches [3]. We present fmcs, which implements a novel multiple MCS algorithm based on subgraph enumeration and subgraph isomorphism testing [4,5] and with algorithm improvements and heuristics which make it competitive to the standard methods. MCS performance evaluation is very sensitive to the test set, so we have developed several reference benchmarks based on ChEMBL-13, including randomly selected pairs of structures, and randomly selected structures with their k = 2, k = 10, and k = 100 nearest neighbors. We use these benchmarks to compare fmcs to SMSD [6] and Indigo’s scaffold detector [7]. Most differences are due to chemistry perception and timeout errors. The fmcs performance, written in Python using the RDKit C++ toolkit [8], is currently between 0.3x and 1.2x the performance of the Indigo implementation in C++. We also cross-validated the fmcs algorithm with the manually curated ChEBI structure ontology classification [9] and characterized the differences. We identified limitations with fmcs, such as with tautomer perception and structural classes that fmcs cannot handle, and problems with ChEBI, such as misclassifications and classifications that are not, structurally speaking, strictly hierarchical.

Stability of an oral ranitidine suspension (15 mg/ml)

PurposeThe aim of this study was to evaluate the microbiological and physicochemical stability of 15 mg/ml ranitidine hydrochloride suspension.Methods15 mg/ml ranitidine hydrochloride suspensions were prepared in commercial vehicle (ORA-BLEND). Suspensions...

Preparation of Modified Cu-Antibacterial White Carbon Black by Microwave Drying

This paper investigated the synthesis and characterization of the Cu-antibacterial White Carbon Black modified by electrolyte, surfactant, and organic solvent. The sol-gel method was used to prepare the White Carbon Black carrier. Cu2+ was selected to be the antibacterial ion, and sodium nitrite (electrolyte), sodium dodecylbenzene sulfonate (surfactant), ethanol (organic solvent) was selected to be the modifiers. They were synthesized on the White Carbon Black carrier. These as-synthesized modified antibacterial materials were characterized by inductively coupled plasma, particle size measurement instrument and enumeration tests (Escherichia coli as experimental bacterium). The result showed that the amount of antibacterial ions and bacteriostasis rate of modified Cu-antibacterial White Carbon Black are higher than those of the common Cu-antibacterial White Carbon Black. In addition, the particle sizes of modified samples can be extended down to 7 µm with a narrow size distribution. Other advantages of modified Cu-antibacterial White Carbon Black are short drying time (only 5 min by microwave), good thermal and light stability.

Synthesis and Characterization of Modified Zn-Antibacterial White Carbon Black

This paper investigated the preparation and characterization of the Zn-antibacterial White Carbon Black modified by electrolyte, surfactant, and organic solvent. The sol-gel method was used to prepare the White Carbon Black carrier. Zn2+ was selected to be the antibacterial ion, and Sodium nitrite (electrolyte), Sodium dodecylbenzene sulfonate (Surfactant), Ethanol (organic solvent) was selected to be the modifiers. They were synthesized on the white carbon black carrier. These as-synthesized modified antibacterial materials were characterized by inductively coupled plasma, particle size measurement instrument and enumeration tests (Escherichia coli as experimental bacterium). The result showed that the amount of antibacterial ions and bacteriostasis rate of modified Zn-antibacterial white carbon black are higher than those for the common Zn-antibacterial white carbon black. In addition, the particle sizes of modified samples can be extended down with a narrow size distribution. Other advantage of modified Zn-antibacterial white carbon black is good thermal stability.

The Mechanism of Antibacterial Activity of Copper and Cerium-Loaded White Carbon Black

This paper investigated the antibacterial mechanism of the Cu-antibacterial White Carbon Black containing cerium. The sol-gel method was used to prepare the White Carbon Black carrier. Cu2+ was selected to be the antibacterial ion, and cerous nitrate was selected to be the additive. They were synthesized on the white carbon black carrier. The structure and antibacterial mechanism of antibacterial material were characterized by inductively coupled plasma, Fourier transform infrared spectroscopy and enumeration tests (Escherichia coli as experimental bacterium). Results showed that the contents of antibacterial ions in the Cu-antibacterial white carbon black containing cerium were higher than those for the general Cu-antibacterial white carbon black (without containing cerium). Cu2+ was bound to white carbon black by ion exchange process. Bacteriostasis rate is over 99%. Furthermore, other advantages of this material are its good thermal and light stabilities.

Study on Antibacterial Mechanism of Ag-Inorganic Antibacterial Material Containing Lanthanum

Inorganic antibacterial materials consist of the antibacterial ions, the additives and the carrier. In this study, we synthesized a new inorganic antibacterial material, of which Ag+ was selected to be the antibacterial ion, lanthanum nitrate served as the additives, and the white carbon black was chosen as the carrier, which was prepared by a sol-gel method. The as-synthesized antibacterial material was characterized by inductively coupled plasma, particle size measurement instrument, and enumeration tests. The result showed that this material has loose and dispersive structure, good thermal and light stability. The possible antibacterial mechanism was also proposed through all the experimental data in this study.

Preparation and Characterization of Cu-Inorganic Antibacterial Material Containing Rare Earths

In this study, we synthesized a new inorganic antibacterial material, of which Cu2+ was selected to be the antibacterial ion, cerous nitrate served as the additives, and the white carbon black was chosen as the carrier, which was prepared by a sol-gel method. The as-synthesized antibacterial material was characterized by inductively coupled plasma, particle size measurement instrument, scanning electron microscope and enumeration tests. The result showed that the amount of antibacterial ions and bacteriostasis rate of this new material are higher than those for the common Cu-inorganic antibacterial material without rare earths. In addition, the particle size of this material can be extended down to 20 μm with a narrow size distribution. Other advantages of this material are its loose and dispersive structure.

Prevalence of tetracycline resistance and genotypic analysis of populations of Escherichia coli from animals, carcasses and cuts processed at a pig slaughterhouse

A Danish pig slaughterhouse was visited in this study to investigate the impact of carcass processing on prevalence of tetracycline-resistant Escherichia coli, and to identify the origins of carcass contaminations with E. coli by assessing genetic diversity of E. coli populations on carcasses. A total of 105 carcasses were sampled at five sequential stages: after stunning, after scalding, after splitting, after cooling and after cutting. Total and tetracycline-resistant E. coli were counted for each sample and tetracycline resistance prevalence per sample was calculated by the fraction of tetracycline-resistant E. coli out of total E. coli. From 15 repeatedly sampled carcasses, 422 E. coli isolates from faeces, stunned carcasses, split carcasses and chilled carcasses were examined by pulse-field gel electrophoresis (PFGE) and tested for antimicrobial susceptibility. The results showed that E. coli counts and the prevalence of tetracycline-resistant E. coli per sample were both progressively reduced after each sampling stage. PFGE analysis showed that E. coli populations from stunned carcasses were highly genetically diverse, compared with those from split carcasses and faeces. Thirteen carcasses (87%) were contaminated with E. coli that were also isolated from faeces of either the same or other pigs slaughtered on the same day; and 80% of stunned carcasses shared the same E. coli PFGE subtypes. The results suggest that some carcass processing steps in the slaughterhouse were effective in reducing both E. coli numbers and the tetracycline resistance prevalence in E. coli on carcasses. Faeces from the same or other pigs slaughtered on the same day were likely to be an important source of E. coli carcass contamination. Combined data from E. coli enumeration, PFGE typing and antimicrobial susceptibility testing suggest that tetracycline-susceptible E. coli probably persisted better compared to resistant ones during the carcass processing.

Feasibility of Rapidly Generating Sufficient Autologous Human Marrow Stromal Cells for Cellular Therapy.

Marrow stromal cells (MSC) are increasingly employed for human cellular therapy protocols. In some cases, it may be advantageous to use autologous MSC, but the feasibility of quickly generating sufficient MSC for autologous therapy has not been established. We investigated the feasibility of rapidly generating large numbers of autologous, early passage, MSC as a prerequisite for the clinical investigation of MSC in human ischemic stroke. Bone marrow (25–35 mL) was collected from 8 healthy volunteers under an IRB-approved protocol; a mononuclear cell (mnc) fraction prepared by ficol density gradient, a portion of the mnc fraction was suspended in low glucose DMEM with 20% pre-screened FBS and plated in T175 culture flasks at 0.2–0.4 × 106 mnc/cm2; maintained at 37 C in a 5% CO2 incubator; non-adherent cells were removed at 48 h and a portion of the cells were passaged when 80% confluent into additional flasks (12–14 days) at 5700 cells/cm2 and MSC were harvested with trypsin at 21 days. The MSC were washed x3 and resuspended in sterile saline for enumeration and suitability testing. MSC were identified according to ISCT criteria (Dominici M, Cytotherapy 2006; 8(4):315), including plastic adherence, morphology, growth characteristics, and phenotype by flow cytometry (CD73, CD90, CD105 positive; CD14, CD34, CD45, HLA-DR negative) and differentiation into adipocytes. The expected total cell yield was calculated based upon actual cell yields minus the volume of cells required for testing based upon a 70 kg patient.Results: In 8 experiments using different donors, a median of 40 (range 20–225) × 106 mnc were available for plating from the original marrow. The median mnc recovery after ficol density gradient was 37% (range 23–130), but the median CFU-F recovery after ficol from the original marrow was 96% (94–96). The calculated MSC yield after primary culture was a median of 25 (10–53) × 106 cells. The calculated yield of MSC after 1st passage at 21 days was a median of 2.1 (0.9–4.4) × 106 MSC/kg (70 kg patient weight), and the median cell viability was 98.5% (92–97.9). At the end of 1st passage all samples yielded negative gram stains and cultures for mycoplasma, bacteria, fungal growth and the median endotoxin level was 0.027 EU/mL (0.02 – 0.10). The viability of the harvested MSC was well maintained in saline at 1 × 106 MSC/mL for 24 h at 4 C (85% +/− 9%) but not after 48 h (37% +/− 9%) or if stored at room temperature. The effects of supplementation with 5% human serum albumin on MSC storage for 24 h at 4 C were variable, ranging from 27% to 95% viability, depending on the albumin manufacturer (3 mfrs; n=4 each).Conclusion: These studies suggest that sufficient MSC that are suitable for autologous human cellular therapy can be rapidly and reliably generated under GTP conditions, that the resultant MSC may be preserved for up to 24 h if stored at 4 C in either saline or pre-screened human serum albumin.

Development of reference materials for microbiological analysis

The reliability of reference materials (RMs) depends on properties such as fitness, robustness, commutability, stability and homogeneity. The development of RMs for microbiological analysis is especially challenged through questions around the stabilisation and recovery of viable cells, the dispersion of precise numbers of cells, matrix effects and, when using molecular techniques, the presence of nucleic acids (e.g. DNA) of dead and live target organisms. However, RMs are indispensable tools for quality control in microbiological analysis. The Institute for Reference Materials and Measurements (IRMM), as part of the European Commission, concentrates its efforts on the development of RMs to support the development, implementation and monitoring of EU legislation. A special focus is given to highly precise RMs for presence/absence and enumeration tests in microbiological food and water analysis. Another group of new RMs certified by the IRMM comprise DNA-based materials to control the identity of micro-organisms in qualitative assays. All of these activities serve to improve quality control in microbiological analysis.

Delineation of a chemical and biological signature for stormwater pollution in an urban river

Stormwater is a major source of Escherichia coli in urban waterways. This study assessed the chemical and bacterial contaminant load in stormwater entering an urban river using chemical analyses, E. coli enumeration and antibiotic resistance testing. Inline monitoring of five major stormwater outfalls revealed that E. coli concentrations can range from 100 to >240,000 CFU 100ml(-1) in stormwater; 15% of the samples contained >240,000 CFU 100 ml(-1). E. coli levels increased from <200 CFU 100ml(-1) to between 1,500 and >20,000 CFU 100ml(-1) in the receiving waters following storm events. Metals and nutrients were significantly elevated (p < 0.05) in inline stormwater samples compared with baseflow river water. Chromium, zinc, lead and total phosphorus were co-ordinately elevated in stormwater, but E. coli levels did not correlate to this chemical signature. E. coli isolates from stormwater and river water samples (n = 2,478) were tested for resistance to 10 antibiotics and compared with over 1,800 isolates from human and non-human sources. The antibiotic resistance frequencies of isolates from human sources were considerably higher than those found in non-human sources. Isolates from human sources were resistant to multiple antibiotics; however, no specific multiple resistance pattern was found. One of five stormwater outfalls tested contained E. coli resistant to antibiotics at frequencies that were not significantly different from those of E. coli isolates from wastewater treatment plant influent (p < 0.05). The results from antibiotic resistance testing suggested that the elevated E. coli levels observed after storm events are a mixture of human and non-human sources. The results of this study demonstrate that there is a considerable E. coli load to receiving waters from stormwater and that sanitary sewage inputs may not be limited to recognized sewer overflows.

Comparison of commercially available Escherichia coli enumeration tests: Implications for attaining water quality standards

Many states are replacing microbiological water quality standards based on “fecal” or thermotolerant coliforms (ThCs) with new standards that employ Escherichia coli as the indicator organism. Implicit in these new standards are assumptions about the equivalence of E. coli enumeration tests and the E. coli levels that will provide protection equivalent to former ThC standards. To investigate these assumptions, E. coli levels in split samples (tests conducted on portions of the same grab sample) collected from small urban streams were determined using enzyme-specific media (Colilert ®, m-ColiBlue24 ®, and nutrient agar with MUG) and compared to levels determined with conventional culture media (m-FC and m-TEC). Although levels observed with all tests were highly correlated, significantly fewer E. coli were enumerated with m-TEC than with enzyme-specific media (paired Student's t-test, α > 99 % ). In addition, E. coli were found to comprise a larger fraction (84–104%, depending on the test) of the total presumptive ThC concentration than that suggested by the United States Environmental Protection Agency (63%). Both of these observations (1) the improvement in E. coli yields observed with enzyme-specific media, and (2) the greater proportion of ThC organisms that are E. coli, indicate that more water quality violations will occur when enzyme-specific media are used for testing than if conventional culture media are used.

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity.

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.

The Presence of Coliform Bacteria in Canadian Pulp and Paper Mill Water Systems — a Cause for Concern?

Abstract A question raised again and again over many decades by pulp and paper mill personnel and local authorities in many countries is, "Does the finding of large numbers of coliform enteric bacteria in the mill water or biotreatment system indicate a real health hazard for the mill workers or the downstream public?" Some of the difficulties in answering this question arise from the widespread misunderstanding of what coliform indicator (enumeration) tests can tell us. Increasing scientific understanding over the last 30 years has greatly altered the coliform indicator tests, the types of bacteria enumerated, and our ability to distinguish harmless from pathogenic (disease-producing) bacteria. This brief review discusses our current understanding of where and when it is appropriate to use coliform indicator assays and the meaning of the results.

Intrinsic bioremediation of trichloroethylene and chlorobenzene: field and laboratory studies

Activities at a former fire training area at Robins Air Force Base in Georgia, USA resulted in contamination of groundwater with a mixture of trichloroethylene (TCE) and chlorobenzene (CB). Results from the field investigation suggest that intrinsic bioremediation process is occurring, which caused the decrease in TCE and CB concentrations, and increase in TCE degradation byproducts [e.g., dichloroethylene isomers (DCEs), vinyl chloride (VC)] concentrations. Contaminated groundwater samples collected from this site were used to conduct microbial enumeration tests, and used as the inocula for microcosm establishment. Results from the microbial enumeration study indicate that methanogenesis was the dominant biodegradation pattern within the source and mid-plume areas, and the aerobic biodegradation process dominated the downgradient area. Laboratory microcosm experiments were conducted to evaluate the feasibility of using CB as the primary substrate to enhance the intrinsic biodegradation of TCE. Microcosm results suggest that CB can serve as the primary substrate (electron donor), and enhance TCE biodegradation to less-chlorinated compounds under both aerobic cometabolism and reductive dechlorination conditions.

Evaluation of the direct viable count method for temperature-stressed Campylobacter coli

Human pathogens, such as campylobacters, transform into the nonculturable state more or less rapidly in the environment. The question arises, whether these cells are still viable. The direct cell count (DVC) method has been used widely for enumeration and viability testing of different bacterial species. The influence of temperature induced bacterial injury on the applicability of the method, however, has not been examined. Two different Campylobacter coli strains were pre-incubated at low temperatures. The necessary nalidixic acid concentration was strain specific, and they became more sensitive to the inhibitory effect of the antibiotic after temperature downshift. The method was not applicable for cultures that had been pre-incubated at intermediate temperatures, although nonculturability had not yet set in at the beginning of the experiments.