The mechanism of carbonate killing of Escherichia coli.

AIMS: To define the mechanism of carbonate killing in Escherichia coli. METHODS AND RESULTS: Sodium carbonate (150 mM) and ethylenediaminetetracetic acid (EDTA, 60 mM) both killed E. coli K-12 when the pH was 8.5, but ammonium chloride (150 mM) was ineffective. EDTA was a 5-fold more potent agent than carbonate, but some of this difference could be explained by ionization. At pH 8.5, only 1.6% of the carbonate is CO(-2), but nearly 100% of the EDTA is EDTA(-2). CONCLUSION: As carbonate and EDTA had similar effects on viability, cellular morphology, protein release and enzymatic activities, the antibacterial activity of carbonate seems to be mediated by divalent metal binding. SIGNIFICANCE AND IMPACT OF THE STUDY: Cattle manure is often used as a fertilizer, and E. coli from manure can migrate through the soil into water supplies. Previous methods of eradicating E. coli were either expensive or environmentally unsound. However, cattle manure can be treated with carbonate to eliminate E. coli, and the cost of this treatment is less than $0.03 per cow per day.

Differences in Escherichia coli culture conditions can have a large impact on the induction of extreme acid resistance.

A recently isolated Escherichia coli strain (3TF4) survived an acid shock that mimicked the low pH of the human gastric stomach (pH 2, 1 h), but this survival was highly influenced by prior growth conditions. Only 0.01% of the stationary phase cells that had been grown anaerobically in a carbonate medium (2 mg glucose and 0.25 mg yeast extract per ml, 40 mm sodium carbonate, final pH 6.5) survived the acid shock, and the survival of exponential phase cultures was even lower (0.0001%). Small amounts of Trypticase (1.5 mg/ml) increased the survival as much as 5000-fold, but cultures that were provided with higher concentrations of Trypticase (7.5 mg/ml) did not reach the stationary phase in 24 h and were more acid sensitive. Sodium acetate (50 mm) also increased acid resistance, and the increased acid shock survival was greater for the cells that had reached the stationary phase (100 versus 1000-fold, respectively). E. coli 3TF4 cultures that had been grown aerobically in Luria broth were already so acid resistant (survivals greater than 40%) that they did not respond to sodium acetate. E. coli 3TF4 cultures that were refrigerated (5 degrees C, 7 days) were nearly as acid resistant as those that were immediately subjected to acid shock (pH 2.0, 1 h).

The effect of various carbonate sources on the survival of Escherichia coli in dairy cattle manure.

Manure slurries (n = 3) prepared from the feces and urine of lactating dairy cattle (1 part urine, 2.2 parts feces, and 6.8 parts distilled water) had an initial pH of 8.6 +/- 0.1; dissolved carbonate concentrations of 48 +/- 4 mm, and Escherichia coli counts of 5.9 +/- 0.7 logs per ml slurry. The pH of untreated slurries declined to pH 7.0 +/- 0.1 by the 10th day of incubation, and the E. coli count increased approximately 10-fold (P < 0.05). When slurries were treated with Na2CO3, K2CO3, NaHCO3 or Na2CO3.NaHCO3 (0 to 16 g/kg slurry), the dissolved carbonates increased in a linear fashion, but only Na2CO3 and K2CO3 (8 g/kg or greater) or Na2CO3.NaHCO3 (16 g/kg) ensured an alkaline pH. Even relatively low concentrations of Na2CO3 or K2CO3 (8 or 12 g/kg) caused a decrease in E. coli viability (P < 0.05), and E. coli could not be detected if 16 g/kg was added (day 5 or 10 of incubation). Na2CO3.NaHCO3 also caused a decrease in E. coli viability, (P < 0.05), but some E. coli (approximately 104 cells per g) were detected on day 10 even if the concentration was 16 g/kg. NaHCO3 did not prevent the decrease in pH or cause a decrease in E. coli numbers (P > 0.05). Calculations based on the Henderson-Hasselbalch equation (pH and dissolved carbonates) indicated that little E. coli killing was noted until the dissolved carbonate anion concentrations (CO3-2) were greater than 1 mm, but bicarbonate anion (HCO3-) concentrations as high as 180 mm did not affect E. coli viability. These results are consistent with the idea that carbonate anion has antimicrobial properties and can kill E. coli in dairy cattle manure.

Practical mechanisms for interrupting the oral-fecal lifecycle of Escherichia coli.

Escherichia coli is a common gut inhabitant, but it is usually out numbered by strictly anaerobic bacteria. When fecal material is exposed to oxygen, fermentation acids can be respired, and E. coli numbers increase. E. coli can survive for long periods of time in feces, but subsequent proliferation is dependent on its ability to re-enter the gastrointestinal tract via contaminated water and food. The oral-fecal lifestyle of E. coli is facilitated by its ability to survive the low pH of the human gastric stomach. Most strains of E. coli do not cause human disease, but some strains produce toxins and other virulence factors. Mature cattle carry E. coli O157:H7 without showing signs of infection, and beef can be contaminated with cattle feces at slaughter. Cattle manure is often used as a fertilizer by the vegetable industry, and E. coli from manure can migrate through the soil into water supplies. Sanitation, cooking and chlorination have been used to combat fecal E. coli, but these methods are not always effective. Recent work indicates that cattle diets can be modified overcome the extreme acid resistance of E. coli. When cattle were fed have for only a few days, colonic volatile fatty acid concentrations declined, pH increased, and the E. coli were no longer able to survive a pH shock that mimicked the human gastric stomach. E. coli in stored cattle manure eventually become highly acid resistant even if the cattle were fed hay, but these bacteria could be killed by sodium carbonate (150 mM, pH 8.5). Because the diet manipulations and carbonate treatments affected E. coli in general rather than specific serotypes, there is an increased likelihood of successful field application.

The physiological and genetic diversity of bovine Streptococcus bovis strains(1).

Laboratory Streptococcus bovis strains and isolates obtained from a steer fed increasing amounts of grain had similar growth characteristics, but they differed in their sensitivity to 2-deoxyglucose (2DG), a non-metabolizable glucose analog. The addition of 2DG decreased both growth rate (0.92+/-0.34 h(-1)) and growth yield (ranging from 25 to 63%), but these differences could not be correlated with diet. However, isolates from a steer fed a 90% grain diet were more prone to 2DG-dependent lysis than those from a hay diet (P<0.001). All S. bovis laboratory strains and isolates had an identical restriction fragment length polymorphism pattern, when their 16S rDNA was digested with HaeIII and HhaI. However, when genomic BOX elements were amplified, 5-12 bands were observed, and the S. bovis isolates and laboratory strains could be grouped into 13 different BOX types. Strains 26 and 581AXY2 had the same BOX type, but the remaining laboratory strains did not form closely related clusters. Strains JB1 and K27FF4 were most closely related to each other. Most of the fresh isolates (24 out of 30) could be grouped into a single cluster (>90% Dice similarity). This cluster contained isolates from all three diets, but it did not have any of the laboratory strains. The majority (90%) of the isolates obtained from the hay-fed steer exhibited the same BOX type. Because more BOX types were observed if grain was added to the diet, it appears that ruminal S. bovis diversity may be a diet-dependent phenomenon.

Invited review: effects of diet shifts on Escherichia coli in cattle.

Escherichia coli O157:H7 is a pathogenic bacterium that causes acute illness in humans, but mature cattle are not affected. E. coli O157:H7 can enter the human food supply from cattle via fecal contamination of beef carcasses at slaughter. Previous attempts to correlate the incidence of E. coli O157:H7 with specific diets or feeding management practices gave few statistically significant or consistent findings. However, recent work indicates that cattle diets may be changed to decrease fermentation acid accumulation in the colon. When fermentation acids accumulate in the colon and pH decreases, the numbers of acid-resistant E. coli increase; acid-resistant E. coli are more likely to survive the gastric stomach of humans. When cattle were fed hay for a brief period (<7 d), acid-resistant E. coli numbers declined dramatically. Other workers have shown that brief periods of hay feeding can also decrease the number of cattle shedding E. coli O157:H7, and a similar trend was observed if cattle were taken off feed and exposed to simulated transport. These observations indicate that cattle feeding management practices may be manipulated to decrease the risk of foodborne illness from E. coli, but further work will be needed to confirm these effects.

Potential effect of cattle diets on the transmission of pathogenic Escherichia coli to humans.

Grain feeding seems to promote the growth and acid resistance of Escherichia coli in fattening beef cattle, and acid-resistant E. coli are more likely to survive the human gastric stomach. When cattle were fed hay for only five days, the number and acid resistance of E. coli decreased dramatically.

Isolation and identification of ruminal methanogens from grazing cattle.

To obtain information on the diversity of ruminal methanogens in grazing animals, three ruminal methanogens from grazing cattle were characterized and identified. Two of the isolates were rod-shaped, with one staining Gram-positive and being non-motile (BRM9), and the other (BRM16) staining Gram-negative and being motile. These isolates grew only on H(2)/CO(2) and formate, and optimally at 38 degrees C and pH 6.5-7.0. The third isolate (CM1) was non-motile, pseudosarcina-shaped, and grew on H(2)/CO(2), acetate, and methyl-containing compounds, with optimal growth at 40 degrees C and pH 6.5. DNA was prepared from the three isolates, and their 16S rRNA genes were sequenced. Phenotypic data and comparisons of nearly complete 16S rDNA sequences showed that BRM9, BRM16, and CM1 are strains of Methanobacterium formicicum, Methanomicrobium mobile, and Methanosarcina barkeri respectively. To the best of our knowledge, this is the first information on ruminal methanogens in cattle maintained under grazing management.

A re-evaluation of the taxonomy of the genus Anaerovibrio, with the reclassification of Anaerovibrio glycerini as Anaerosinus glycerini gen. nov., comb. nov., and Anaerovibrio burkinabensis as Anaeroarcus burkinensis [corrig.] gen. nov., comb. nov.

Chemotaxonomic, electron microscopic and 16S rRNA gene sequence analyses of the three described species of the genus Anaerovibrio demonstrated only remote similarities to each other. The 16S rRNA gene sequence similarities between Anaerovibrio lipolytica, Anaerovibrio glycerini and Anaerovibrio burkinabensis and the derived phylogenetic relationships of the three species studied fell below genus level. All three species clustered within the Sporomusa-Pectinatus-Selenomonas phyletic group. Each species showed a distinct phospholipid pattern and whole-cell fatty acid distribution. Several isoprenologues of the lipoquinone 'lipid F' were found to differ in their quantitative distribution in the Anaerovibrio species. On the basis of these results, the new genera Anaerosinus gen. nov. and Anaeroarcus gen. nov. are proposed. The type species of Anaerosinus is Anaerosinus glycerini comb. nov., and the type species of Anaeroarcus is Anaeroarcus burkinensis [corrig.] comb. nov. The genus Anaerovibrio is consequently restricted to a single species, namely Anaerovibrio lipolyticus [corrig.]

Isolation and characterization of two glycerol-fermenting clostridial strains from a pilot scale anaerobic digester treating high lipid-content slaughterhouse waste.

Two obligately anaerobic bacterial strains were isolated from the contents of a pilot scale, anaerobic digester treating slaughterhouse waste with a high protein and lipid content. The isolates, LIP1 and MW8, were characterized as spore-forming, Gram-positive rods, capable of fermenting glycerol. Isolate LIP1 was also observed to be lipolytic and was able to hydrolyse tallow and olive oil. Both isolates grew optimally at 37 degrees C and formed either acetate and formate (LIP1), or acetate and butyrate (MW8), as major glycerol fermentation products. Both isolates produced ethanol as the major reduced fermentation end-product. Neither MW8 nor LIP1 had growth and metabolism inhibited by the addition of stearic acid at concentrations normally considered bactericidal. Analysis of the 16S rRNA gene sequences, in conjunction with the phenotypic data, confirmed that the isolates are members of the genus Clostridium (sensu lato), clustering with species of clostridial clusters I (MW8) and XIVa (LIP1).

Isolation and characterisation of obligately anaerobic, lipolytic bacteria from the rumen of red deer.

Two Gram-positive, obligately anaerobic, lipolytic bacteria, isolates LIP4 and LIP5, were obtained from the rumen contents of juvenile red deer. These mesophilic bacterial strains were capable of hydrolysing the neutral lipids, tallow, tripalmitin and oliver oil, into their constituent free long-chain fatty acid and glycerol moieties. The latter compound was dissimilated by both isolates, with isolate LIP4 producing propionate as the predominant product, while isolate LIP5 produced acetate, ethanol and succinate. The lactate-utilising isolate LIP4 grew on a limited range of saccharide substrates including glucose, fructose and ribose, and exhibited an unusual cell wall structure and morphology. The isolate LIP5 grew upon a wider range of saccharides, but was unable to use lactate as a substrate. Based upon phenotypic and 16S rRNA gene sequence analyses, isolate LIP4 clusters with species in the genus Propionibacterium, while isolate LIP5 is a member of clostridial cluster XIVa.

Formate and ethanol are the major products of glycerol fermentation produced by a Klebsiella planticola strain isolated from red deer.

The rumen contents of red deer (Cervus elaphus) were used to isolate bacterial capable of fermenting glycerol. The biochemistry, physiology, morphology and phylogeny of one isolate were studied in detail. The isolate (DR3) was tentatively identified as a strain of the species Klebsiella planticola as based on phenotypic characterization. The data obtained from 16S rRNA sequence analysis showed that the deer rumen isolate DR3 was 99.7% similar to the type strain of Kl. planticola (DSM 3069T), thus confirming the results of the phenotypic characterization. During cell growth, it was established that glycerol dissimilation by Kl. planticola DR3 led to the production of formate and ethanol at equimolar levels of 32 mmol 1(-1) and 30 mmol 1(-1), respectively. As a result of the data obtained, a closed carbon balance was constructed for Kl. planticola DR3. This finding represented the first report of the complete end-product profile for glycerol dissimilation by a strain of Kl. planticola isolated from cervine rumen contents.