Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and Curli production on its resistance to chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log(10) CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 microg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12 degrees C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Antimetabolite sensitivity and magnesium uptake by thermally stressed Vibrio parahaemolyticus

Metabolic inhibitors were added to a culture medium inoculated with theramlly stressed Vibrio parahaemolyticus to obtain information pertaining to biosynthetic processes required for recovery from heat damage. Ribonucleic acid and protein syntheses, in addition to membrane repair, were required during recovery of injured cells. Neither nucleic acid nor Mg2+ leakage was noted to occur during the time cells were subjected to heat stress. Studies revealed that Mg2+ was apparently taken up by cells of V. parahaemolyticus during the first 30 min after thermal treatment, indicating a possible increased requirement for Mg2+ for membrane and/or ribosome stability and repair.     

Pregnancy outcomes in women with repeated implantation failures after intracytoplasmic morphologically selected sperm injection (IMSI)

Background

The role of serum anti-Müllerian hormone (AMH) as predictor of in-vitro fertilization outcomes has been much debated. The aim of the present study is to investigate the practicability of combining serum AMH level with biological age as a simple screening method for counseling IVF candidates of advanced reproductive age with potential poor outcomes prior to treatment initiation.

Methods

A total of 1,538 reference patients and 116 infertile patients aged greater than or equal to 40 years enrolled in IVF/ICSI cycles were recruited in this retrospective analysis. A reference chart of the age-related distribution of serum AMH level for Asian population was first created. IVF/ICSI patients aged greater than or equal to 40 years were then divided into three groups according to the low, middle and high tertiles the serum AMH tertiles derived from the reference population of matching age. The cycle outcomes were analyzed and compared among each individual group.

Results

For reference subjects aged greater than or equal to 40 years, the serum AMH of the low, middle and high tertiles were equal or lesser than 0.48, 0.49-1.22 and equal or greater than 1.23 ng/mL respectively. IVF/ICSI patients aged greater than or equal to 40 years with AMH levels in the low tertile had the highest cycle cancellation rate (47.6%) with zero clinical pregnancy. The nadir AMH level that has achieved live birth was 0.56 ng/mL, which was equivalent to the 36.4th percentile of AMH level from the age-matched reference group. The optimum cut-off levels of AMH for the prediction of nonpregnancy and cycle cancellation were 1.05 and 0.68 ng/mL, respectively.

Conclusions

Two criteria: (1) age greater than or equal to 40 years and (2) serum AMH level in the lowest tertile (equal or lesser than 33.3rd percentile) of the matching age group, may be used as markers of futility for counseling IVF/ICSI candidates.     

Human pathogens associated with raw produce and unpasteurized juices, and difficulties in decontamination

The ability of public health agencies to identify, through enhanced epidemiologic and surveillance techniques, raw fruits, vegetables, and unpasteurized juices as probable sources of infectious microorganisms, has undoubtedly resulted in increased numbers of documented outbreaks. Changes in agronomic, harvesting, distribution, processing, and consumption patterns and practices have also likely contributed to this increase. The risk of illness associated with raw produce and unpasteurized produce products can be reduced by controlling or preventing contamination, or by removing or killing pathogenic microorganisms by washing or treating them with sanitizers. However, the hydrophobic cutin, diverse surface morphologies, and abrasions in the epidermis of fruits and vegetables limit the efficacy of these treatments. Journal of Industrial Microbiology & Biotechnology (2001) 27, 104–110. Received 23 June 2000/ Accepted in revised form 17 October 2000     

Incidence of airborne Aspergillus flavus spores in cornfields of five states.

Airborne Aspergillus flavus spores were rarely detected throughout the 1975 growing season by the agar plate method at cornfield sites in Georgia, Illinois, Maryland, South Carolina, and Virginia.     

Effectiveness of Disinfectants in Killing Enterobacter sakazakii in Suspension, Dried on the Surface of Stainless Steel, and in a Biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Influence of modified-atmosphere storage on the growth of uninjured and heat-injured Aeromonas hydrophila.

The growth of uninjured and heat-injured Aeromonas hydrophila incubated at 5 degrees C (22 days) and 30 degrees C (31 h) under air, N2, and CO2 was investigated. At 30 degrees C, the growth patterns of cells on brain heart infusion agar incubated under air and N2 were similar, although slight differences in the lengths of the lag phases and the final populations were detected. The lag phases of cells incubated under air and N2 were substantially longer at 5 degrees C than at 30 degrees C. The population of uninjured A. hydrophila incubated at 5 degrees C under air and N2 remained constant, whereas the number of injured cells declined before the exponential growth phase. Growth at 5 degrees C was enhanced when uninjured and heat-injured A. hydrophila were incubated under N2. At 30 degrees C, cells incubated under CO2 exhibited noticeably longer lag phases and lower growth rates than did cells incubated under air and N2. The viable populations of uninjured and heat-injured cells incubated at 5 degrees C under CO2 declined steadily throughout incubation.     

Combined effects of water activity, temperature and chemical treatments on the survival of Salmonella and Escherichia coli O157:H7 on alfalfa seeds

AIMS: The objective of this study was to determine the combined effects of water activity (a(w)), chemical treatment and temperature on Salmonella and Escherichia coli O157:H7 inoculated onto alfalfa seeds. METHODS AND RESULTS: Alfalfa seeds inoculated with Salmonella or E. coli O157:H7 and adjusted to various a(w) values were subjected to simultaneous and separate treatments with chemicals and heat. The rate of death of both pathogens was correlated with increased a(w) (0.15-0.60) and temperature (5-37 degrees C) over a 52-week storage period. Higher seed a(w) enhanced the inactivation of pathogens on seeds heated at 50-70 degrees C for up to 24 h. Treatment of seeds with water, 1% Ca(OH)2, 1% Tween 80, 1% Ca(OH)2 plus 1% Tween 80 or 40 mg l(-1) Tsunami 200 at 23 or 55 degrees C for 2 min significantly (alpha=0.05) reduced populations of Salmonella and E. coli O157:H7. CONCLUSIONS: Overall, at the combinations of temperature and concentrations of chemicals tested, 1% Ca(OH)2 was most effective in killing Salmonella and E. coli O157:H7 without reducing seed viability. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the treatments evaluated in this study, whether applied separately or in combination, eliminated Salmonella or E. coli O157:H7 on alfalfa seeds without sacrificing the viability of the seeds. It remains essential that practices to prevent the contamination of alfalfa seeds be strictly followed in order to minimize the risk of Salmonella and E. coli O157:H7 infections associated with sprouts produced from these seeds.     

Tolerance of acid-adapted and non-adapted Escherichia coli O157:H7 cells to reduced pH as affected by type of acidulant

A study was carried out to determine if three strains of Escherichia coli O157:H7 grown (18 h) in Tryptic Soy Broth (TSB) and TSB supplemented with 1.25% glucose (TSBG), i.e. unadapted and acid-adapted cells, respectively, exhibited changes in tolerance to reduced pH when plated on Tryptic Soy Agar (TSA) acidified (pH 3.9, 4.2, 4.5, 4.8, 5.1 and 5.4) with acetic, citric or malic acids. All test strains grew well on TSA acidified with acetic acid at pH > or = 5.4 or malic acid at pH > or = 4.5; two strains grew on TSA acidified with citric acid at pH > or = 4.5, while the third strain grew at pH > or = 4.8. Acid-adapted and control (unadapted) cells differed little in their ability to form visible colonies on TSA containing the same acid at the same pH. However, on plates not showing visible colonies, acid-adapted cells retained higher viability than unadapted cells when plated on acidified TSA. Growth of acid-adapted and control cells of E. coli O157:H7 inoculated into TSB containing acetic acid (pH 5.4 and 5.7) and citric or malic acids (pH 4.2 and 4.5) was also studied. There was essentially no difference in growth characteristics of the two types of cells in TSB acidified at the same pH with a given acid. Tolerance of acid-adapted and control cells on subsequent exposure to low pH is influenced by the type of acidulant. The order of sensitivity at a given pH is acetic > citric > malic acid. When performing acid challenge studies to determine survival and growth characteristics of E. coli O157:H7 in foods, consideration should be given to the type of acid to which cells have been exposed previously, the procedure used to achieve acidic environments and possible differences in response among strains. The use of strains less affected by pH than type of acidulant or vice versa could result in an underestimation of the potential for survival and growth of E. coli O157:H7 in acid foods.     

Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration

Introduction

The degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.

Methods

Quantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.

Results

Initial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.

Conclusions

The release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.