Factors that inhibit the amplification of nucleic acids by PCR are present with target DNAs from many sources. The inhibitors generally act at one or more of three essential points in the reaction in the following ways: they interfere with the cell lysis necessary for extraction of DNA, they interfere by nucleic acid degradation or capture, and they inhibit polymerase activity for amplification of target DNA. Although a wide range of inhibitors is reported, the identities and modes of action of many remain unclear. These effects may have important implications for clinical and public health investigations, especially if the investigations involve food and environmental screening. Common inhibitors include various components of body fluids and reagents encountered in clinical and forensic science (e.g., hemoglobin, urea, and heparin), food constituents (e.g., organic and phenolic compounds, glycogen, fats, and Ca 21 ), and environmental compounds (e.g., phenolic compounds, humic acids, and heavy metals). Other, more widespread inhibitors include constituents of bacterial cells, nontarget DNA and contaminants, and laboratory items such as pollen, glove powder, laboratory plasticware, and cellulose. This review discusses the findings of many studies related to clinical, food, and environmental microbiology, including approaches that have been used to overcome inhibition and facilitate amplification for detection and typing. Few areas of biological science remain untouched by the invention of PCR (34, 81, 99). Other methods for amplifying nucleic acids (72, 123), such as Qb replicase (18), ligase chain reaction (13, 128), single-stranded sequence replication (17, 47), strand displacement amplification (126, 127), and nucleic acid sequence-based amplification (23, 122), have been described, but these methods have received less attention. Problems sometimes occur with PCR, however (124). Despite early indications of great sensitivity, the sensitivity of PCR may be a negative aspect of the procedure, since the most commonly reported problem is false-positive results due to cross-contamination (98, 124). This problem can be overcome by UV irradiation (100), with sodium hypochlorite (92), and by photochemical or enzymic methods (25, 36, 40, 78). One problem that is less discussed is reaction inhibition. This may be total or partial and can manifest itself as complete reaction failure or as reduced sensitivity of detection. In some cases, inhibition may be the cause of false-negative reactions, since few workers incorporate internal controls in each reaction tube. Early evidence of exquisite sensitivity with mammalian cells (53) involving detection of a single molecule of DNA from a hair was not reproduced when PCR was applied to many microbial (and some mammalian) situations, where poor sensitivity, specificity, and reproducibility have been reported (16, 82, 86, 129, 132, 134). There may also be potentially important effects in PCR typing reactions (121), and difficulties can occur in post-PCR manipulation (61). Although systematic study of inhibition has seldom been the focus of published investigations, many workers have reported these effects in the course of other studies (12, 19, 21, 124, 129, 132, 133). Considering the prevalence of this problem, it is surprising that few systematic and mechanistic studies of PCR inhibition have been reported. Rossen et al. (97) contributed the most comprehensive study of PCR inhibition, identifying inhibitory factors in foods, bacterial culture media, and various chemical compounds. These inhibitory factors included organic and inorganic chemicals, detergents, antibiotics, buffers, enzymes, polysaccharides, fats, and proteins. This review lists and discusses inhibitors and methods that can overcome the attenuation of amplification in clinical, food, and environmental microbiology. It is beyond the scope of this paper to discuss in detail the various physical, enzymic, and chemical methods used in the extraction, purification, and quantitation of nucleic acids. Those methods are presented and discussed in commercial literature and elsewhere (14, 95, 96, 106, 134).

https://aem.asm.org/content/aem/63/10/3741.full.pdf

Inhibition and facilitation of nucleic acid amplification.

Summary: Over the past decade, the genus Aeromonas has undergone a number of significant changes of practical importance to clinical microbiologists and scientists alike. In parallel with the molecular revolution in microbiology, several new species have been identified on a phylogenetic basis, and the genome of the type species, A. hydrophila ATCC 7966, has been sequenced. In addition to established disease associations, Aeromonas has been shown to be a significant cause of infections associated with natural disasters (hurricanes, tsunamis, and earthquakes) and has been linked to emerging or new illnesses, including near-drowning events, prostatitis, and hemolytic-uremic syndrome. Despite these achievements, issues still remain regarding the role that Aeromonas plays in bacterial gastroenteritis, the extent to which species identification should be attempted in the clinical laboratory, and laboratory reporting of test results from contaminated body sites containing aeromonads. This article provides an extensive review of these topics, in addition to others, such as taxonomic issues, microbial pathogenicity, and antimicrobial resistance markers.

The Genus Aeromonas: Taxonomy, Pathogenicity, and Infection

http://capridairyworld.org/files/pdf/RUMIN-physico-chem-published.pdf

Physico-chemical characteristics of goat and sheep milk

Listeria monocytogenes is a Gram positive, aerobic, facultative anaerobic and nonacid fast bacterium, which can cause the disease listeriosis in both human and animals. It is widely distributed thoroughout the environment and has been isolated from various plant and animal food products associated with listeriosis outbreaks.

Contaminated ready-to-eat food products such as gravad and cold-smoked salmon and rainbow trout have been associated with human listeriosis in Sweden. The aim of this study was to analyse the occurrence and level of L. monocytogenes in
gravad and cold-smoked salmon (Salmo salar) products packed under vacuum or modified atmosphere from retail outlets in Sweden. Isolated strains were characterized by serotyping and the diversity of the strains within and between
producers were determined with PFGE (Pulsed-field gel electrophoresis). The characterized fish isolates were compared with previously characterized human strains.
L. monocytogenes was isolated from 11 (three manufacturers) of 56 products analysed. This included gravad salmon products from three manufacturers and cold-smoked salmon from one manufacturer. The highest level of L. monocytogenes found was 1500 cfu/g from a cold-smoked salmon product but the level was low (<100 cfu/g) in most of the products. Serovar 1/2a was predominant,
followed by 4b. Three products of gravad salmon harboured more than one serovar. PFGE typing of the 56 salmon isolates detected five Asc I types: four types were identical to human clinical strains with Asc I and one was identical and one was closely related to human clinical strains with Apa I. Isolation of identical or closely related L. monocytogenes strains from human clinical cases of listeriosis and gravad and cold-smoked salmon suggested that these kinds of products are possible sources of listeriosis in Sweden. Therefore, these products should be considered risk products for human listeriosis.

Listeria monocytogenes, a food-borne pathogen

It is well known that the presence of lactobacilli is important for the maintenance of the intestinal microbial ecosystem (39). They have been shown to possess inhibitory activity toward the growth of pathogenic bacteria such as Listeria monocytogenes (3, 25, 36, 42), Escherichia coli, Salmonella spp. (8, 16, 27), and others (4, 13, 37). This inhibition could be due to the production of inhibitory compounds such as organic acids, hydrogen peroxide, bacteriocins (30), or reuterin (4) or to competitive adhesion to the epithelium. In order to survive in and colonize the gastrointestinal tract, probiotic bacteria should express high tolerance to acid and bile and have the ability to adhere to intestinal surfaces (31, 34). Survival in and temporary colonization of the human gastrointestinal tract have been demonstrated for some lactic acid bacteria (1, 23, 29). However, in vivo testing is expensive and time consuming and requires approval by ethical committees. Therefore, reliable in vitro methods for selection of promising strains are required.

Enterocyte-like Caco-2 cells (38) have been successfully used for in vitro studies on the mechanism of cellular adhesion of nonpathogenic lactobacilli (10, 24, 40, 43) and bifidobacteria (5, 15). Moreover, this cell line has been used to examine the mechanism of cellular adhesion and invasion of pathogenic bacteria such as L. monocytogenes (21), Salmonella typhimurium (20), and E. coli (32). Recently, Caco-2 cells have been used to examine the antimicrobial activity of lactobacilli (6, 13, 27) and bifidobacteria (5) against pathogenic bacteria. Antimicrobial properties of lactobacilli have been determined by using three methods: inhibitory activity toward the growth of test bacteria in vitro (7, 13, 14), inhibitory activity toward cell association, and invasion of pathogens using cultured human intestinal cells (6, 7, 12–14, 27), as well as protection of conventional or germfree mice against bacterial infection (7, 13, 14, 27). These showed how antimicrobial activities observed by in vitro methods could be confirmed in vivo as well.

In the present study, the Caco-2 cell line was used to study the adhesive properties of 47 potentially probiotic cultures in vitro. The cultures were also examined for antimicrobial properties toward pathogenic bacteria along with tolerance to low pH and bile salts. Among these cultures, five promising strains were examined by in vivo studies. The abilities of the selected strains to survive passage through the gastrointestinal tract and maintain colonization was tested in fecal samples using API 50CHL and internal transcribed spacer PCR (ITS-PCR) for primary selection of strains and restriction enzyme analysis (REA) combined with pulsed-field gel electrophoresis (PFGE) for confirmation of isolates recovered from fecal samples during and after administration. It was the main objective of this study to compare the in vitro evaluation of certain properties of various Lactobacillus spp. that are important for their survival in the gastrointestinal tract with their actual ability to survive in vivo.

https://aem.asm.org/content/aem/65/11/4949.full.pdf

Screening of Probiotic Activities of Forty-Seven Strains of Lactobacillus spp. by In Vitro Techniques and Evaluation of the Colonization Ability of Five Selected Strains in Humans

Considerable knowledge has been accumulated on the biochemical processes occurring during ripening of Cheddar cheese, which in turn has major consequences on flavor and texture development. The present review outlines major metabolic pathways and agents involved in the modification of milk constituents in Cheddar cheese ripening. Mechanisms of volatile flavor and off-flavor production and recent developments in the analysis, both sensory and instrumental, of Cheddar flavor and flavor compounds are also detailed here.

/pdf/flavor-of-cheddar-cheese-a-chemical-and-sensory-perspective-1dwze8j5ek.pdf

Flavor of Cheddar Cheese: A Chemical and Sensory Perspective

Invited Review: Sensory Analysis of Dairy Foods

: Escherichia coli encounter numerous different stresses during their growth, survival, and infection. These stresses are relevant to survival in foods and food processing environments. E. coli and other bacteria respond to stress conditions by activating small or large groups of genes under the control of common regulator proteins. Stress conditions result in the accumulation of these regulator proteins and subsequent transcription of many genes allows cells to cope with specific stress situations, conferring stress tolerance and survival. In addition, induced stress tolerance of cells is attributed to enhanced virulence and enhanced tolerance to other stresses (cross-protection). In this review, regulation of stress and the stress tolerance response of E. coli to heat, acid, starvation, and cold stresses that are commonly used in food preservation and food production will be addressed. The effect of different stress on survival, adaptation, and cross-protection of E. coli studied using laboratory media, and real foods will be briefly summarized. Finally, the relationship of stress response and subsequent virulence and cross-protection will also be discussed.

Stress Response of Escherichia coli


A standardized descriptive language for Cheddar cheese flavor was developed and validated. Representative Cheddar cheeses (240) were collected. Fifteen individuals from industry, academia, and government participated in a 3-d roundtable discussion to generate descriptive flavor terms. A highly trained descriptive panel (n = 11) refined the terms and identified references. Cheddar cheeses (24) were presented to the panel for validation with the identified lexicon. The panel differentiated the 24 Cheddar cheeses as determined by univariate and multivariate analysis of variance (P < 0.05). Twenty-seven terms were identified to describe Cheddar flavor. Seventeen descriptive terms were present in most Cheddar cheeses. A standard sensory language for Cheddar cheese will facilitate training and communication between different research groups.

/pdf/development-of-a-descriptive-language-for-cheddar-cheese-4y7dku3xj5.pdf

Development of a Descriptive Language for Cheddar Cheese

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens, Escherichia coli O157:H7 and enterotoxigenic Staphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37°C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1,400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121°C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80°C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

MaryAnne Drake

Papers

Flavor of Cheddar Cheese: A Chemical and Sensory Perspective

Invited Review: Sensory Analysis of Dairy Foods

Stress Response of Escherichia coli

Development of a Descriptive Language for Cheddar Cheese

rRNA Stability in Heat-Killed and UV-Irradiated Enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7†