Lethality of a heat- and phosphate-catalyzed glucose by-product to Escherichia coli O157:H7 and partial protection conferred by the rpoS regulon.
A by-product of glucose produced during sterilization (121 degrees C, 15 lb/in2, 15 min) at neutral pH and in the presence of phosphate (i.e., phosphate-buffered saline) was bactericidal to Escherichia coli O157:H7 (ATCC 43895). Other six-carbon (fructose and galactose) and five-carbon (arabinose, ribose, and xylose) reducing sugars also produced a toxic by-product under the same conditions. Fructose and the five-carbon sugars yielded the most bactericidal activity. Glucose concentrations of 1% (wt/vol) resulted in a 99.9% decline in the CFU of stationary-phase cells per milliliter in 2 days at 25 degrees C. An rpoS mutant (pRR10::rpoS) of strain 43895 (FRIK 816-3) was significantly (P < 0.001) more sensitive to the glucose-phosphate by-product than the parent strain, as glucose concentrations from 0.05 to 0.25% resulted in a 2- to 3-log10 reduction in CFU per milliliter in 2 days at 25 degrees C. Likewise, log-phase cells of the wild-type strain, 43895, were significantly more sensitive (P < 0.001) to the glucose-phosphate by-product than were stationary-phase cells, which is consistent with the stability of rpoS and the regulation of rpoS-regulated genes. The bactericidal effect of the glucose-phosphate by-product was reduced when strains ATCC 43895 and FRIK 816-3 were incubated at a low temperature (4 degrees C). Also, growth in glucose-free medium (i.e., nutrient broth) did not alleviate the sensitivity to the glucose-phosphate by-product and excludes the possibility of substrate-accelerated death as the cause of the bactericidal effect observed. The glucose-phosphate by-product was also bactericidal to Salmonella typhimurium, Shigella dysenteriae, and a Klebsiella sp. Attempts to identify the glucose-phosphate by-product were unsuccessful. These studies demonstrate the production of a glucose-phosphate by-product bactericidal to E. coli O157:H7 and the protective effects afforded by rpoS-regulated gene products. Additionally, the detection of sublethally injured bacteria may be compromised by the presence of this by-product in recovery media.
Byrd
J J
JJ
Department of Biology, St. Mary's College of Maryland, St. Mary's City, Maryland 20686, USA. JJByrd@osprey.smcm.edu
Cheville
A M
AM
Bose
J L
JL
Kaspar
C W
CW
D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D002021:Buffers; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D006358:Hot Temperature; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D018085:Regulon; D012808:Sigma Factor / Q000235:genetics* / Q000502:physiology; D012965:Sodium Chloride / Q000737:chemistry; D013242:Sterilization / Q000379:methods
D001426:Bacterial Proteins; D002021:Buffers; D010710:Phosphates; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D012965:Sodium Chloride; D005947:Glucose