Biomedical subjects

Francisco Diez-Gonzalez

Publications and source records attributed to Francisco Diez-Gonzalez.

12 recordsLinked to original sources

PubMed2006

Modeling the growth of Listeria monocytogenes based on a time to detect model in culture media and frankfurters.

In the current study, temperature dependent growth of Listeria monocytogenes strain H7776, with an initial inoculum level of approximately 0.1 CFU/mL or g, was modeled based on "time to detect" (TTD) using the Arrhenius equation. The activation energies (E(a)) obtained from specific growth rate and lag phase duration in tryptic soy broth (TSB) and frankfurters were compared. At 4 degrees C, the TTD for L. monocytogenes was 217 h in TSB and 48 h in portions of frankfurters. The TTD decreased as temperature increased from 4 to 36 degrees C in both culture media and frankfurters, and this relationship was modeled using the Arrhenius equation. Based on this model, the E(a) values for the TTD in TSB and frankfurters averaged 22.7 and 18.7 kcal/mol, respectively, and were not significantly different (p<0.05). Linear regression was performed on the exponential part of the growth curve to evaluate specific growth rate constants at each temperature using the Monod model. The E(a) values were also calculated based on the specific growth rate and the lag phase of L. monocytogenes in TSB and frankfurters incubated in the same range of temperatures. The average E(a) values for the specific growth rates and the lag phase durations in TSB cultures were 19 and 21 kcal/mol, respectively. In frankfurters, the average E(a) values were slightly greater for both specific growth rate and lag phase duration (29 and 35 kcal/mol, respectively), but these values were not significantly different from the E(a) calculated for the TTD in each medium. These results indicate that the TTD concept can be used to develop and validate safety-based shelf life models.

Colony Count, Microbial↗

PubMed2006

Prevalence of shiga toxin-encoding bacteria and shiga toxin-producing Escherichia coli isolates from dairy farms and county fairs.

Shiga toxin-encoding bacteria (STB) and shiga toxin-producing Escherichia coli (STEC) were detected and isolated from dairy cattle and their farm environment and from manure piles at Minnesota (MN) county fairs from 2001 to 2002. A total of 2,540 samples were collected from 28 dairy cattle farms (8 organic and 20 conventional), 17 calf pens (5 organic and 12 conventional), and 12 county fairs. STB were detected from 71 (3.2%) of 2208 fecal samples with 20 (71.4%) of 28 dairy farms having at least one positive animal sample. In samples collected from conventional farms, 41 (2.3%) of 1750 fecal samples were STB-positive and 13 (65%) of 20 farms had at least one positive animal. Thirty (6.6%) of 458 fecal samples from organic farms were STB-positive and 7 (87.5%) of 8 farms had at least one positive animal. STB was detected from 31 (17.4%) of 178 samples and 7 (58.3%) out of 12 manure piles at county fairs. A total of 43 STEC isolates were recovered and belonged to 26 different serotypes (19 O and 18 H types). Among STEC, 60.5% possessed only stx1, 30.2% stx2, and 9.3% both stx1 and stx2. The genes eae and hlyA were detected in more than 50% of the STEC isolates. STB can be found on most dairy cattle farms including organic and conventional herds and county fairs. The presence of these potentially pathogenic bacteria in county fairs may pose a risk to the public who have contact with cattle or their environment.

Animals↗

PubMed2006

Thermal inactivation of Bacillus anthracis spores in cow's milk.

Decimal reduction time (time to inactivate 90% of the population) (D) values of Bacillus anthracis spores in milk ranged from 3.4 to 16.7 h at 72 degrees C and from 1.6 to 3.3 s at 112 degrees C. The calculated increase of temperature needed to reduce the D value by 90% varied from 8.7 to 11.0 degrees C, and the Arrhenius activation energies ranged from 227.4 to 291.3 kJ/mol. Six-log-unit viability reductions were achieved at 120 degrees C for 16 s. These results suggest that a thermal process similar to commercial ultrahigh-temperature pasteurization could inactivate B. anthracis spores in milk.

Animals↗

PubMed2006

Prevalence and characterization of Escherichia coli O157 isolates from Minnesota dairy farms and county fairs.

Samples were collected from 26 organic and conventional farms and 12 county fairs in Minnesota during 2001 and 2002 to identify the presence of Escherichia coli O157. Immunomagnetic separation was used for isolation of E. coli O157. Isolates were further characterized by the presence of virulence marker genes (stx1, stx2, eaeA, E-hly, katP, etpD, and espP), antimicrobial susceptibility profiles, and genotypes. During 2001, E. coli O157 was isolated from 16 (5.2%) of 305 fecal samples and from 7 (36.8%) of 19 farms. During 2002, E. coli O157 was isolated from 6 (4.5%) of 132 fecal samples from weaned calves at 4 (23.5%) of 17 farms. During 2001 and 2002, cattle manure samples were collected from 12 county fairs, and E. coli O157 was isolated from 19 (11%) of 178 samples and 9 (75%) of 12 county fairs. Among 40 E. coli O157 isolates, 17 isolates (43%) had both the stx1 and stx2 genes, and 21 strains (53%) had the stx2 gene only. Thirteen percent of O157 isolates were resistant to tetracycline, and 25% were resistant to sulfadimethoxine. Heterogeneity of E. coli O157 strains was demonstrated by the presence of 22 different pulsed-field gel electrophoresis (PFGE) patterns. Four PFGE patterns matched those of isolates previously found in humans. The presence of E. coli O157 at county fairs suggests the potential for transmission to the public, who may have contact with cattle or their environment.

Animals↗

PubMed2006

Longitudinal microbiological survey of fresh produce grown by farmers in the upper midwest.

Microbiological analyses of fruits and vegetables produced by farms in Minnesota and Wisconsin were conducted to determine coliform and Escherichia coli counts and the prevalence of E. coli, Salmonella, and E. coli O157:H7. During the 2003 and 2004 harvest seasons, 14 organic farms (certified by accredited organic agencies), 30 semiorganic farms (used organic practices but not certified), and 19 conventional farms were sampled to analyze 2,029 preharvest produce samples (473 organic, 911 semiorganic, and 645 conventional). Produce varieties included mainly lettuces, leafy greens, cabbages, broccoli, peppers, tomatoes, zucchini, summer squash, cucumber, and berries. Semiorganic and organic farms provided the majority of leafy greens and lettuces. Produce samples from the three farm types had average coliform counts of 1.5 to 2.4 log most probable number per g. Conventional produce had either significantly lower or similar coliform populations compared with the semiorganic and organic produce. None of the produce samples collected during the 2 years of this study were contaminated with Salmonella or E. coli O157:H7. E. coli contamination was detected in 8% of the samples, and leafy greens, lettuces, and cabbages had significantly higher E. coli prevalence than did all the other produce types in both years for the three farm types. The prevalence of E. coli contamination by produce type was not significantly different between the three farm types during these 2 years, with the exception of organic leafy greens, in which E. coli prevalence was one-third that of semiorganic leafy greens in 2003. These results indicate that the preharvest microbiological quality of produce from the three types of farms was very similar during these two seasons and that produce type appears to be more likely than farm type to influence E. coli contamination.

Agriculture↗

PubMed2004

A novel glutamate-dependent acid resistance among strains belonging to the Proteeae tribe of Enterobacteriaceae.

Morganella, Providencia and Proteus strains were capable of surviving pH 2.0 for 1 h if glutamate was present. These strains did not have glutamic acid decarboxylase activity and the gadAB genes were not detected in any of these bacteria. When exposed to pH 2.0 acid shocks, the survival rate of these bacteria was significantly increased with glutamate concentrations as low as 0.3 mM in the acid media. Escherichia coli cells incubated at pH 3.4 consumed four times more glutamate and produced at least 7-fold more gamma-amino butyric acid than Morganella, Providencia and Proteus strains. These results indicate that strains belonging to the Proteeae tribe might have novel glutamate dependent acid-resistance mechanisms.

Enterobacteriaceae↗

PubMed2004

Characterization of an E2-type colicin and its application to treat alfalfa seeds to reduce Escherichia coli O157:H7.

Several outbreaks of Escherichia coli O157:H7 infections have been associated with contaminated alfalfa seeds. A recently isolated E. coli strain Hu194 was capable of inhibiting 22 strains of E. coli O157:H7 and this inhibition was mediated by the production of a colicin named Hu194. The objectives of this study were to test the efficacy of treating alfalfa seeds with colicin Hu194 against E. coli O157:H7 strains, and to characterize this antimicrobial protein. Significant reductions (approximately 5 log CFU ml-1) in the viable cell counts of strains 43890 and 43895 were observed after 1-day incubation with semi-crude colicin, and after 2 days for strain 3081. Strain 43890 was successfully eliminated (5 log CFU g-1) from inoculated alfalfa seeds after soaking in a colicin suspension at a concentration of 10,000 AU/g. Treatment of alfalfa seeds inoculated with strains 43895 and 3081 required 20-fold higher concentrations of colicin Hu194 to achieve as much as 3 log CFU g-1 reductions. The genes encoding the colicin Hu194 operon were located on a 6 kb plasmid, and the sequence analysis revealed that this colicin was an E-type DNAse. From the sequence data, the estimated molecular masses of colicin Hu194, its immunity protein and lysis protein were 61.3, 10.0 and 4.8 kDa, respectively. Based on DNA and protein sequence comparisons with other E-type colicin, colicin Hu194 belonged to the type E2-colicin cluster. However, cross-immunity tests between E-group colicins suggested that Hu194 colicin was divergent from the previously characterized E2 colicins.

Bacteriocin Plasmids↗

PubMed2004

Reduction of Escherichia coli O157:H7 populations in cattle by addition of colicin E7-producing E. coli to feed.

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10(7) CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log(10) CFU/g was observed, with a maximum decrease of 1.8 log(10) CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10(8) CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.

Animal Feed↗

PubMed2004

Characterization of colicinogenic Escherichia coli strains inhibitory to enterohemorrhagic Escherichia coli.

A previously identified set of anti-Escherichia coli O157:H7 colicinogenic E. coli were characterized to assess the suitability of these isolates as a preharvest food safety intervention in cattle. This collection of 23 E. coli strains were screened for virulence factors, antibiotic resistance, type of colicin(s) present, and their ability to inhibit other pathogenic E. coli. With the use of PCR, pathogen genes were detected in six of the 23 colicinogenic E. coli. When the nonpathogenic strains were assessed for antibiotic resistance, four strains showed resistance to at least one antibiotic. The remaining set of 14 strains were evaluated for the presence of previously identified colicins. Seven colicins (B, El, E2/E7, E7, Ia/Ib, K, and M) were detected. One half of the strains possessed multiple types of colicins. The most commonly detected colicins were B, E2/E7, and M, which were found in six strains each. DNA sequencing was also performed in order to classify the E2/E7 colicins separately from E7 colicins. The 14 colicinogenic E. coli also were evaluated for their ability to inhibit 10 different non-O157 pathogenic E. coli. Six of the colicinogenic E. coli were capable of inhibiting all 10 pathogens, and the remaining eight strains could each inhibit between six to eight of the pathogenic E. coli. This strain collection has great potential for inhibiting E. coli O157:H7 in cattle.

Animals↗

PubMed2004

Preharvest evaluation of coliforms, Escherichia coli, Salmonella, and Escherichia coli O157:H7 in organic and conventional produce grown by Minnesota farmers.

Microbiological analyses of fresh fruits and vegetables produced by organic and conventional farmers in Minnesota were conducted to determine the coliform count and the prevalence of Escherichia coli, Salmonella, and E. coli O157:H7. A total of 476 and 129 produce samples were collected from 32 organic and 8 conventional farms, respectively. The samples included tomatoes, leafy greens, lettuce, green peppers, cabbage, cucumbers, broccoli, strawberries, apples, and seven other types of produce. The numbers of fruits and vegetables was influenced by their availability at participating farms and varied from 11 strawberry samples to 108 tomato samples. Among the organic farms, eight were certified by accredited agencies and the rest reported the use of organic practices. All organic farms used aged or composted animal manure as fertilizer. The average coliform counts in both organic and conventional produce were 2.9 log most probable number per g. The percentages of E. coli-positive samples in conventional and organic produce were 1.6 and 9.7%, respectively. However, the E. coli prevalence in certified organic produce was 4.3%, a level not statistically different from that in conventional samples. Organic lettuce had the largest prevalence of E. coli (22.4%) compared with other produce types. Organic samples from farms that used manure or compost aged less than 12 months had a prevalence of E. coli 19 times greater than that of farms that used older materials. Serotype O157:H7 was not detected in any produce samples, but Salmonella was isolated from one organic lettuce and one organic green pepper. These results provide the first microbiological assessment of organic fruits and vegetables at the farm level.

Escherichia coli↗

PubMed2002

Selection of recently isolated colicinogenic Escherichia coli strains inhibitory to Escherichia coli O157:H7.

Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E. coli per animal species ranged from 14% for horse isolates to 64% for sheep strains. Those isolates that inhibited E. coli K12 were screened against E. coli O157:H7, and 42 strains were found to be capable of inhibiting all 22 pathogenic strains tested. None of these 42 strains produced bacteriophages, and only 24 isolates inhibited serotype O157:H7 in liquid culture. The inhibitory activity of these strains was completely eliminated by treatment with proteinase K. When mixtures of these 24 colicinogenic strains were grown in anaerobic continuous culture, the four-strain E. coli O157:H7 population was reduced at a rate of 0.25 log10 cells per ml per h, which was fivefold faster than the washout rate. Two strains originally isolated from cat feces (F16) and human feces (H30) were identified by repetitive sequences polymerase chain reaction as the predominant isolates in continuous cultures. The results of this work indicate that animal species other than cattle can be sources of anti-O157 colicinogenic strains, and these results also lead to the identification of at least two isolates that could potentially be used in preharvest control strategies.

Animals↗

PubMed1997

The ability of Escherichia coli O157:H7 to decrease its intracellular pH and resist the toxicity of acetic acid.

Batch cultures of Escherichia coli K-12 grew well in an anaerobic glucose medium at pH 5.9, but even small amounts of acetate (20 mM) inhibited growth and fermentation. E. coli O157:H7 was at least fourfold more resistant to acetate than K-12. Continuous cultures of E. coli K-12 (pH 5.9, dilution rate 0.085 h-1) did not wash out until the sodium acetate concentration in the input medium was 80 mM, whereas E. coli O157:H7 persisted until the sodium acetate concentration was 160 mM. E. coli K-12 cell accumulated as much as 500 mM acetate, but the intracellular acetate concentration of O157:H7 was never greater than 300 mM. Differences in acetate accumulation could be explained by intracellular pH and the transmembrane pH gradient (delta pH). E. coli K-12 maintained a more or less constant delta pH (intracellular pH 6.8), but E. coli O157:H7 let its delta pH decrease from 0.9 to 0.2 units as sodium acetate was added to the medium. Sodium acetate increased the rate of glucose consumption, but there was little evidence to support the idea that acetate was creating a futile cycle of protons. Increases in glucose consumption rate could be explained by increases in D-lactate production and decreases in ATP production. Intracellular acetate was initially lower than the amount predicted by delta pH, but intracellular acetate and delta pH were in equilibrium when the external acetate concentrations were high. Based on these results, the acetate tolerance of O157:H7 can be explained by fundamental differences in metabolism and intracellular pH regulation. By decreasing the intracellular pH and producing large amounts of D-lactate, O157:H7 is able to decrease delta pH and prevent toxic accumulations of intracellular acetate anion.

Acetic Acid↗