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M W Griffiths

Publications and source records attributed to M W Griffiths.

At least 19 recordsLinked to original sources
PubMed2006

Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K.

AIMS: To understand the potential use of bacteriophage K to treat bovine Staphylococcus aureus mastitis, we studied the role of whey proteins in the inhibition of the phage-pathogen interaction in vitro. METHODS AND RESULTS: The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in raw bovine whey and serum. Incubation of S. aureus with phage in whey showed that the bacteria are more resistant to phage lysis when grown in whey and also bovine serum. Whey collected from 23 animals showed a wide variation in the level of phage-binding inhibition. The role of the protein component of milk whey in this inhibition was established; treatment of the whey by heat, proteases and ultrafiltration removed the inhibitory activity. Brief exposure of S. aureus cells to whey, followed by resuspension in broth, also reduced phage binding. Microscopy showed the adhesion of extracellular material to the S. aureus cell surface following exposure to whey. Chromatographic fractionation of the whey demonstrated that the inhibitory proteins were present in the high molecular weight fraction. CONCLUSIONS: The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phage attachment and thereby hindered lysis. The inhibitory whey proteins are of high molecular weight in their native form and may sterically block phage attachment sites on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have implications for any future use of phage therapy in the treatment of mastitis, and other diseases, caused by S. aureus. This pathogen is predicted to be much more resistant to phage treatment in vivo than would be expected from in vitro broth culture experiments.

Animals↗
PubMed2006

Efficacy and pharmacokinetics of bacteriophage therapy in treatment of subclinical Staphylococcus aureus mastitis in lactating dairy cattle.

Bovine mastitis is an inflammation of the udder caused by microbial infection. Mastitis caused by Staphylococcus aureus is a major concern to the dairy industry due to its resistance to antibiotic treatment and its propensity to recur chronically. Growing concerns surrounding antibiotic resistance have spurred research into alternative treatment methods. The ability of lytic S. aureus bacteriophage K to eliminate bovine S. aureus intramammary infection during lactation was evaluated in a placebo-controlled, multisite trial. Twenty-four lactating Holstein cows with preexisting subclinical S. aureus mastitis were treated. Treatment consisted of 10-ml intramammary infusions of either 1.25 x 10(11) PFU of phage K or saline, administered once per day for 5 days. The cure rate was established by the assessment of four serial samples collected following treatment. The cure rate was 3 of 18 quarters (16.7%) in the phage-treated group, while none of the 20 saline-treated quarters were cured. This difference was not statistically significant. The effects of phage intramammary infusion on the bovine mammary gland were also studied. In healthy lactating cows, a single infusion of either filter-sterilized broth lysate or a CsCl gradient-purified phage preparation elicited a large increase in the milk somatic cell count. This response was not observed when phage was infused into quarters which were already infected with S. aureus. Phage-infused healthy quarters continued to shed viable bacteriophage into the milk for up to 36 h postinfusion. The phage concentration in the milk suggested that there was significant degradation or inactivation of the infused phage within the gland.

Animals↗
PubMed2006

Role of efflux pumps in adaptation and resistance of Listeria monocytogenes to benzalkonium chloride.

In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC.

Adaptation, Physiological↗
PubMed2006

Major advances in fresh milk and milk products: fluid milk products and frozen desserts.

Major technological advances in the fluid milk processing industry in the last 25 yr include significant improvements in all the unit operations of separation, standardization, pasteurization, homogenization, and packaging. Many advancements have been directed toward production capacity, automation, and hygienic operation. Extended shelf-life milks are produced by high heat treatment, sometimes coupled with microfiltration or centrifugation. Other nonthermal methods have also been investigated. Flavored milk beverages have increased in popularity, as have milk beverages packaged in single-service, closeable plastic containers. Likewise, the frozen dairy processing industry has seen the development of large-capacity, automated processing equipment for a wide range of products designed to gain market share. Significant advancements in product quality have been made, many of these arising from improved knowledge of the functional properties of ingredients and their impact on structure and texture. Incidents of foodborne disease associated with dairy products continue to occur, necessitating even greater diligence in the control of pathogen transmission. Analytical techniques for the rapid detection of specific types of microorganisms have been developed and greatly improved during this time. Despite tremendous technological advancements for processors and a greater diversity of products for consumers, per capita consumption of fluid milk has declined and consumption of frozen dairy desserts has been steady during this 25-yr period.

Animals↗
PubMed2006

Occurrence of proteolytic activity and N-acyl-homoserine lactone signals in the spoilage of aerobically chill-stored proteinaceous raw foods.

Proteolytic pseudomonads dominate the spoilage flora of aerobically chill-stored proteinaceous raw foods. Proteolysis during spoilage of these food systems affects both food quality and the dynamics of the bacterial community because it increases the availability of nutrients to the community as a whole. Quorum sensing, or cell-cell signaling, is associated closely with ecological interactions among bacteria in mixed communities. The potential role of quorum sensing in proteolytic food spoilage was examined, based on the evaluation of N-acyl-homoserine lactone (AHL) signal molecules. The occurrence of proteolytic activity and AHL signals was studied during spoilage of aerobically chill-stored ground beef, fish, chicken, and raw milk. Pseudomonads dominated the psychrotrophic flora, followed distantly by members of the Enterobacteriaceae. The growth of pseudomonads was correlated with the occurrence of proteolytic activity in all food systems. AHL concentration began increasing significantly only after the onset of proteolytic activity. Widely divergent AHL profiles were revealed by thin-layer chromatography analysis of the different food samples, and these profiles were likely determined by the undefined bacterial flora in these systems and by the characterized pseudomonads and Enterobacteriaceae. Although Hafnia alvei was a major component of the Enterobacteriaceae flora in all foods tested and a strong AHL producer, the signal molecules produced by H. alvei strain EB1 did not influence protease production by Pseudomonas fluorescens strain 395 in vitro. These results do not indicate any clear correlation between the overall detectable AHL signal molecules accumulated in the food samples and proteolytic activity.

4-Butyrolactone↗
PubMed2005

Microbiological sampling of poultry carcass portions by excision, rinsing, or swabbing.

Groups of 25 skin-on thighs or skin-on, skinned, or tumbled breast portions of broiler chicken carcasses were sampled by excision of skin or muscle tissue, rinsing, or swabbing. Counts of total aerobic bacteria, coliforms, and Escherichia coli were recorded for each sample. For all types of carcass portions, the mean log counts and the total log counts obtained for each group of bacteria by excision or rinsing mostly differed by <0.5 log unit. However, the counts obtained by swabbing were generally >0.5 log unit lower than the smaller of the values obtained by the other two sampling methods.

Animals↗
PubMed2005

Comparison of methods for the isolation of thermotolerant Campylobacter from poultry.

Human campylobacteriosis has become the major cause of foodborne gastroenteritis in industrialized countries. Although there have been numerous studies investigating the prevalence of Campylobacter in animals and raw meat, sensitive and low-cost detection methods are needed to implement effective control measures during primary production and to use as tools in risk assessment studies. Thermophilic Campylobacter spp. in naturally contaminated (n = 64) and inoculated (n = 16) broiler samples were detected using two International Organization for Standardization (ISO)-approved methods. Both enrichment broths (those of Preston and of Park and Sanders) were inoculated with (i) Campylobacter jejuni ATCC35921, (ii) boneless breast from broilers, (iii) boneless breast rinse solution, (iv) boneless breast rinse solution inoculated with C. jejuni ATCC35921 before centrifugation, and (v) boneless breast rinse solution inoculated with C. jejuni ATCC35921 after centrifugation. The results indicated that the Park and Sanders broth was superior to the Preston broth for recovery of Campylobacter spp., and no significant differences (P > 0.05) were found between ISO (meat pieces) and modified ISO (centrifuged chicken rinse solution) methods for the detection of Campylobacter spp.

Animals↗
PubMed2003

Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence.

Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

Adenosine Triphosphate↗
PubMed2002

Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells.

AIMS: To develop methods to assess the efficiency of immunomagnetic separation (IMS). METHODS AND RESULTS: The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of <or= 105 cfu ml-1, in a 1-ml sample volume, nearly 80-85% recovery of the pathogen was observed after 0.5 h of incubation. For an 11-ml sample containing 104 cfu ml-1, maximum recovery (50% of cells) was achieved only after 2 h incubation. CONCLUSIONS: The detection limit of an ATP-based bioluminescent assay for E. coli O157:H7 was reduced by 1 log cycle after optimization of IMS. The bioluminescent methods could be used for screening and testing the affinity of antibodies or other affinity elements of biosorbents towards live bacterial cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Bioluminescent assays provide an easy way to optimize conditions for the capture of bacteria by biosorbents in real time.

Adenosine Triphosphate↗
PubMed2001

Effect of environmental and chemotactic stimuli on the activity of the Campylobacter jejuni flaA sigma(28) promoter.

The effect of environmental and chemotactic stimuli was assessed using a fusion between the Campylobacter jejuni flaA sigma(28) promoter and a promoterless luxCDABE cassette on pRYluxCDABE. Environmental stimuli relevant to the enteric environment were found to modulate flaA promoter activity. pH, bovine bile, deoxycholate, L-fucose and osmolarity up-regulated the flaA promoter while viscosity resulted in down-regulation. C. jejuni chemotactic effectors, including the amino acids aspartate and glutamate and the organic acids citrate, fumarate, alpha-ketoglutarate and succinate up-regulated the flaA promoter. Proline, which does not elicit a chemotactic response, up-regulated the flaA promoter.

Animals↗
PubMed2001

Detection of Salmonella and simultaneous detection of Salmonella and Shiga-like toxin-producing Escherichia coli using the magnetic capture hybridization polymerase chain reaction.

The magnetic capture hybridization polymerase chain reaction (MCH-PCR) was used to detect Salmonella and also to simultaneously detect Salmonella and Shiga-like toxin-producing Escherichia coli (SLTEC). Fifty-seven Salmonella and 41 SLTEC were included in the study. Salmonella were detected either individually by a single MCH-PCR assay targeting the inv gene or simultaneously with SLTEC by a multiplex MCH-PCR in which SLTEC were detected using primers for the slt genes. Both single and multiplex assays were found to be specific for tested pathogens. The results indicate that MCH-PCR can be used as means of detecting single or multiple bacterial pathogen(s).

Adhesins, Bacterial↗
PubMed2001

Influence of phage population on the phage-mediated bioluminescent adenylate kinase (AK) assay for detection of bacteria.

AIMS: The effect of phage concentration on the activity of adenylate kinase (AK) released from the cells lysed during infection was investigated in order to optimize a bioluminescent phage-mediated method for bacterial enumeration. METHODS AND RESULTS: The number of bacteria lysed by phages specific to Salmonella enteritidis and E. coli was determined using a bioluminescent method for the detection of AK released. In order to optimize the assay, the effect of phage concentration and time of infection on the amount of AK released was investigated. The release of AK was greatest at a multiplicity of infection (moi) of 10-100. CONCLUSION: The amount of AK released from Salmonella enteritidis and E. coli G2-2 cells by specific phages, SJ2 and AT20, respectively, depended on the type of bacteria, the stage of growth, the nature of phage, moi and time. SIGNIFICANCE AND IMPACT OF THE STUDY: An assay is described which allows detection of E. coli and Salmonella Enteritidis within 2 h at levels of 103 cfu ml-1.

Adenylate Kinase↗
PubMed2001

Development and optimization of a novel immunomagnetic separation- bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth.

Salmonella is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of Salmonella in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of Salmonella enterica serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10(4) CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of Salmonella serovar Enteritidis in broth cultures which can be applied to preenriched food samples.

Bacteriological Techniques↗
PubMed2001

Use of luminescent Campylobacter jejuni ATCC 33291 to assess eggshell colonization and penetration in fresh and retail eggs.

A luminescent phenotype in Campylobacter jejuni ATCC 33291, generated by a transcriptional fusion between the C. jejuni flaA sigma28 promoter and the luxCDABE genes of Xenorhabdus luminescens on plasmid pRYluxCDABE, was used to examine colonization and penetration of fresh and retail eggs. C. jejuni colonized both fresh and retail eggs at 37, 40, and 42 degrees under microaerophilic conditions. Fresh eggs were more heavily colonized than retail eggs. Under aerobic conditions, fresh eggs were colonized at similar levels for all three temperatures. C. jejuni was found to penetrate the eggshell in 2 of 48 (4.2%) fresh eggs assessed. Although the lux+ phenotype did not provide an effective means of predicting penetration sites, it was effective at visualizing eggshell colonization. Also, it effectively demonstrated the organism's opportunistic nature, as eggshell surfaces with flaws and slight cracks were extensively colonized and easily detected by a photon counting charge-coupled device camera. Using scanning electron microscopy, C. jejuni ATCC 33291 was visualized on both the exterior and interior surfaces of the egg membranes indicating penetration of these barriers.

Animals↗
PubMed2001

Modeling the survival of Escherichia coli O157:H7 in uncooked, semidry, fermented sausage.

The data collected from studies to monitor inactivation of Escherichia coli O157:H7 in uncooked fermented salami were used to develop models to describe survival of the organism. Three models were developed that included different variables to best describe E. coli O157:H7 reduction. Model A included the variables water activity (a(w)), pH, time, and quadratic variables pH and time. Model B separated the processing stages into fermentation and drying. The fermentation included the variables pH and temperature x time (ttarea) and interaction between the two variables. The drying stage was modeled using the variables time and a(w) and interaction between the two. Model C looked at variables a(w) and the time at pH 5.3 to achieve a 2-D log reduction of E. coli O157:H7 and the interaction between the variables. The variables selected for inclusion in all the models were significant at the P < 0.0001 level. The predicted values for all models correlated well to the observed values with R2 of 0.888, 0.828, 0.836, and 0.818 for models A. Bferm, Bdrying, and C, respectively. The models were validated using data from a trial not used to develop the model. In general the predicted reduction in E. coli O157:H7 count in uncooked fermented salami was greater than for the observed E. coli O157:H7 log reductions for all models, but the relation between the two was linear. The results demonstrate that modeling can be a useful tool in assessing manufacturing practices for uncooked fermented salami processes.

Animals↗
PubMed2001

Effect of phage on survival of Salmonella enteritidis during manufacture and storage of cheddar cheese made from raw and pasteurized milk.

The ability of Salmonella Enteritidis to survive in the presence of phage, SJ2, during manufacture, ripening, and storage of Cheddar cheese produced from raw and pasteurized milk was investigated. Raw milk and pasteurized milk were inoculated to contain 10(4) CFU/ml of a luminescent strain of Salmonella Enteritidis (lux) and 10(8) PFU/ml SJ2 phage. The milks were processed into Cheddar cheese following standard procedures. Cheese samples were examined for Salmonella Enteritidis (lux), lactic acid bacteria, molds and yeasts, coliforms, and total counts, while moisture, fat, salt, and pH values were also measured. Salmonella Enteritidis (lux) was enumerated in duplicate samples by surface plating on MacConkey novobiocin agar. Bioluminescent colonies of Salmonella Enteritidis were identified in the NightOwl molecular imager. Samples were taken over a period of 99 days. Counts of Salmonella Enteritidis (lux) decreased by 1 to 2 log cycles in raw and pasteurized milk cheeses made from milk containing phage. In cheeses made from milks to which phage was not added, there was an increase in Salmonella counts of about 1 log cycle. Lower counts of Salmonella Enteritidis (lux) were observed after 24 h in pasteurized milk cheese containing phage compared to Salmonella counts in raw milk cheese with phage. Salmonella Enteritidis (lux) survived in raw milk and pasteurized milk cheese without phage, reaching a final concentration of 10(3) CFU/g after 99 days of storage at 8 degrees C. Salmonella did not survive in pasteurized milk cheese after 89 days in the presence of phage. However, Salmonella counts of approximately 50 CFU/g were observed in raw milk cheese containing phage even after 99 days of storage. In conclusion, this study demonstrates that the addition of phage may be a useful adjunct to reduce the ability of Salmonella to survive in Cheddar cheese made from both raw and pasteurized milk.

Animals↗
PubMed2001

Reduction in levels of Escherichia coli O157:H7 in apple cider by pulsed electric fields.

Many studies have demonstrated that high voltage pulsed electric field (PEF) treatment has lethal effects on microorganisms including Escherichia coli O157:H7; however, the survival of this pathogen through the PEF treatment is not fully understood. Fresh apple cider samples inoculated with E. coli O157:H7 strain EC920026 were treated with 10, 20, and 30 instant charge reversal pulses at electric field strengths of 60, 70, and 80 kV/cm, at 20, 30, and 42 degrees C. To accurately evaluate the lethality of apple cider processing steps, counts were determined on tryptic soy agar (TSA) and sorbitol MacConkey agar (SMA) to estimate the number of injured and uninjured E. coli O157:H7 cells after PEF treatment. Cell death increased significantly with increased temperatures and electric field strengths. A maximum of 5.35-log10 CFU/ml (P < 0.05) reduction in cell population was achieved in samples treated with 30 pulses and 80 kV/cm at 42 degrees C. Cell injury measured by the difference between TSA and SMA counts was found to be insignificant (P > 0.05). Under extreme conditions, a 5.91-log10 CFU/ml reduction in cell population was accomplished when treating samples with 10 pulses and 90 kV/cm at 42 degrees C. PEF treatment, when combined with the addition of cinnamon or nisin, triggered cell death, resulting in a reduction in E. coli O157:H7 count of 6 to 8 log10 CFU/ml. Overall, the combination of PEF and heat treatment was demonstrated to be an effective pasteurization technique by sufficiently reducing the number of viable E. coli O157:H7 cells in fresh apple cider to meet U.S. Federal Drug Administration recommendations.

Beverages↗