Claudia Balagué

Effect of 2,4-dichlorophenoxyacetic acid herbicide on Escherichia coli growth, chemical composition, and cellular envelope

2,4‐Dichlorophenoxyacetic acid (2,4‐D) is a herbicide widely used in the world and mainly excreted by the renal route in exposed humans and animals. Herbicides can affect other nontarget organisms, such as Escherichia coli. We observed that a single exposure to 1 mM 2,4‐D diminished growth and total protein content in all E. coli strains tested in vitro. In addition, successive exposures to 0.01 mM 2,4‐D had a toxic effect decreasing growth up to early stationary phase. Uropathogenic E. coli adhere to epithelial cells mediated by fimbriae, adhesins, and hydrophobic properties. 2,4‐D exposure of uropathogenic E. coli demonstrated altered hydrophobicity and fimbriation. Hydrophobicity index values obtained by partition in p‐xylene/water were 300–420% higher in exposed cells than in control ones. Furthermore, values of hemagglutination titer, protein contents in fimbrial crude extract, and electron microscopy demonstrated a significant diminution of fimbriation in treated cells. Other envelope alterations could be detected, such as lipoperoxidation, evidenced by decreased polyunsaturated fatty acids and increased lipid degradation products (malonaldehyde), and motility diminution. These alterations decreased cell adherence to erythrocytes, indicating a diminished pathogenic capacity of the 2,4‐D‐exposed E. coli. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 43–53, 2001

Phage biocontrol of enteropathogenic and shiga toxin-producing Escherichia coli in meat products.

Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were selected based on their host ranges, and their lytic effects on pathogenic E. coli strains inoculated on pieces of beef were determined. We evaluated the reductions of viable cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat after exposure to DT6 at 5 and 24°C for 3, 6, and 24 h and the effect of both phages against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7 × 1010 plaque forming units per milliliter (PFU/mL) for DT1 and 1.4 × 1010 PFU/mL for DT6, the most effective. When enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested, we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after 3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast, all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5 × 10−7–1.8 × 10−6) and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, presented lytic activity for different E. coli virotypes and could be useful in reducing Shiga toxigenic E. coli and enteropathogenic E. coli viable cells in meat products.

Effect of the herbicide 2,4-dichlorophenoxyacetic acid on uropathogenic Escherichia coli virulence factors.

The effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-widely used in the world and mainly excreted by the renal route in exposed humans-were studied on the virulence and surface characteristics of an uropathogenic Escherichia coli strain. When the urine was supplemented with 2,4-D in vitro, the compound significantly reduced the bacterial fimbriation assayed by hemagglutination and surface protein quantification. Protein values decreased from 0.24 mg/g dw to 0.05 or 0.12 mg/g dw by 1 or 0.1 mM 2,4-D treatment, respectively. The effects in vivo were studied in groups of mice challenged intra-urethra with E. coli and exposed by the oral route with three different 2,4-D doses (2.6, 25 or 70 mg/kg bw) during 22 days. Depending on the dose used, the herbicide significantly decreased or removed bacterial cells in mice bladder and kidneys; except in the group treated with the highest dose from the 9th day of treatment. The histological studies showed mononuclear cell infiltration at low doses, and toxic damage in the renal parenchyma at prolonged exposure with higher doses, up to tisular necrosis in the 70 mg/kg bw group after 9 days of treatment. Our investigations performed in an experimental model suggest that short time 2,4-D exposure at low doses could act in prevention of UTI stimulating leukocytic migration and decreasing bacterial fimbriation. On the contrary, high doses and long-term exposure enhanced renal damage resulting in infection recurrence.

Effect of preservatives on Shiga toxigenic phages and Shiga toxin of Escherichia coli O157:H7.

Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through the induction of the integrated bacteriophages that encode the toxin genes. These phages might be the principal means for the dissemination and release of Shiga toxins. We evaluated the effect of three common food preservatives, potassium sorbate, sodium benzoate, and sodium propionate, on the propagation of the phages and Shiga toxins. We tested each preservative at four concentrations, 1, 1.25, 2.5, and 5 mg/ml, both on free phages and on lysogenic phages in bacteria. We also evaluated the expression of a lambdoid phage, which was exposed to increasing concentrations of preservatives, by measuring β-galactosidase activity from SPC105, a transductant strain. Furthermore, we tested the effect of the preservatives on cytotoxigenic activity of Shiga toxin on Vero cells. We detected an increase of the inhibitory effect of the phage lytic activity, both in lysogenic and free phages, as the preservative concentration increased. However, the inhibition was higher on the lysogenic phages release than on free phages. Sodium benzoate and potassium sorbate were about equal at inhibiting phages; they were more effective than sodium propionate. A significant decrease of lacZ expression, encoded in a lambda phage, was observed. We also found a reduction in Shiga toxin titer caused by exposure of E. coli O157:H7 to 5 mg/ml sodium benzoate or potassium sorbate. These results imply that these three preservatives, used to inhibit microbial spoilage of foods, also act to inhibit lytic activity and dispersion of a phage carrying the gene encoding powerful Shiga cytotoxins. Also notable was the inactivation of Shiga toxin activity, although this effect was detected using concentrations of preservatives greater than those allowed by the Argentine Food Code.

Inhibition of the adhesion to catheters of uropathogenic Escherichia coli by sub-inhibitory concentrations of cefotaxime.

OBJECTIVE To investigate the effect of sub-inhibitory concentrations of cefotaxime on adherence to siliconized latex urinary catheters of uropathogenic Escherichia coli strains from pregnant and non pregnant patients. STUDY DESIGN Using random sampling, 30 E. coli strains were selected from hospitalized patients with catheter associated urinary tract infection, 12 from pregnant women and 18 from men and non-pregnant women. The strains were categorized on the basis of cefotaxime susceptibility, adhesion and biofilm production capacity, cell surface hydrophobicity and expression of adhesins and fimbriae in vitro. RESULTS The overall results indicated that sub-inhibitory concentrations of cefotaxime could reduce the adhesiveness, the biofilm production and hence, potentially, the infection rate associated with indwelling urinary catheters. CONCLUSION Based on our results, we propose that this reduction is due to decreasing exopolysaccharide production and increasing cell surface hydrophobicity of E.coli strains.

Resistance of phages lytic to pathogenic Escherichia coli to sanitisers used by the food industry and in home settings

Fil: Tomat, David Damian. Universidad Nacional de Rosario. Facultad de Ciencias Bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina

Evaluation of a novel cocktail of six lytic bacteriophages against Shiga toxin-producing Escherichia coli in broth, milk and meat

Phages are potentially useful as antimicrobial agents in food, especially cocktails of different phages which may prevent the development of bacterial resistance. Biocontrol assays with a six-phage cocktail, which is lytic against DH5α, an enteropathogenic (EPEC) and two Shiga-toxigenic (STEC) Escherichia coli strains, were performed in Hershey-Mg broth, milk and meat at refrigerated (4 °C), room (24 °C) and abusive (37 °C) temperatures. At 4 °C, cell counts were significantly lower (2.2-2.8 log10 CFU/mL) when E. coli strains (∼109 CFU/mL) were challenged against the phage cocktail (∼109 PFU/mL) in Hershey-Mg broth after 24 h. However, reductions were higher (3.2-3.4 log10 CFU/mL) after a 48 h exposure for all the strains tested. In addition, reduction values reached up to 3.4 log10 CFU/mL (24 °C) and 3.6 log10 CFU/mL (37 °C) in challenge tests after 24 h, though the reductions achieved were slightly lower after 48 h for the four E. coli strains tested. In milk, the cocktail was highly effective since bacterial counts were below the detection limit (<101 CFU/mL) at 4 °C, while the reductions ranged from 2 to 4 log10 CFU/mL at 24 °C after a 24 h exposure. At 37 °C, DH5α was eliminated within 2 h, and an average cell decrease of 4 log10 CFU/mL was observed for the three pathogenic strains tested. When the assays were performed in meat, biocontrol values ranged from 0.5 to 1.0 log10 CFU/mL after 48 h at 4 °C, while a higher cell inactivation was achieved at 24 °C (2.6-4.0 log10 CFU/mL) and 37 °C (3.0-3.8 log10 CFU/mL). Furthermore, higher inactivation values for O157:H7 STEC (1.55 ± 0.35 log10 CFU/mL) at 4 °C were obtained in meat when incubation was extended up to 6 days. As a conclusion, our six-phage cocktail was highly effective at 24 °C and 37 °C, though less effective at 4 °C in both food matrices evaluated. Thus, it might be applied against pathogenic EPEC and STEC strains to prevent foodborne diseases especially when the cold chain is lost.