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Spread and change in stress resistance of Shiga toxin-producing Escherichia coli O157 on fungal colonies.

Lee K, Kobayashi N, Watanabe M, Sugita-Konishi Y, Tsubone H, Kumagai S, Hara-Kudo Y - Microb Biotechnol (2013)

Bottom Line: To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin-producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food-related fungi including fermentation starters.The population of STEC O157 increased when co-cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus.After co-culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

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D values at pH 2.5 of (A) motile and (B) non-motile strains of STEC O157 after co-culture with various fungi.Approximately 108 CFU of motile or non-motile STEC O157 was inoculated onto a 7-day-old fungal colony grown on PDA. As control, the same amount of STEC O157 was inoculated onto PDA without fungi. In addition, to investigate the effect of a filamentous structure per se on the stress resistance of STEC O157, the bacterium was inoculated onto a sterilized rectangular cotton wool (width 20 × depth 40 × height 4 mm) that was placed onto PDA as ‘control with cotton wool’. After 7-day incubation at 25°C, the agar strip was crushed and suspended in PBS. The suspension was mixed at a full speed by using an automatic mixer and centrifuged at 4000 × g for 10 min. The supernatant was removed, and the pellet washed twice with PBS. The pellet was re-suspended again with PBS and was diluted 100-fold into 10 ml of EG medium acidified with hydrogen chloride at pH 2.5. EG medium is commonly used in evaluating acid resistance of E. coli (Lin et al., 1996). The broth was incubated at 37°C, and the population of STEC O157 was measured at 0, 1, 2 and 4 h. To enumerate STEC O157, the inoculated broth was serially diluted with PBS and pour-plated onto TSA. After incubation for 48 h at 37°C, colonies were counted. D values were calculated using the formula (D value = −1/slope), where slope represents the linear regression of the data including all the sampling points. The R2 values of the linear regression analyses were more than 0.8 in every analysis. The experiments were performed in triplicate. Error bars represent the standard deviation of the three trials. Each letter in the figures represents a significant difference by Student's t-test as follows:A. P < 0.05 compared to the control.B. P < 0.05 compared to the control with cotton wool.
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fig02: D values at pH 2.5 of (A) motile and (B) non-motile strains of STEC O157 after co-culture with various fungi.Approximately 108 CFU of motile or non-motile STEC O157 was inoculated onto a 7-day-old fungal colony grown on PDA. As control, the same amount of STEC O157 was inoculated onto PDA without fungi. In addition, to investigate the effect of a filamentous structure per se on the stress resistance of STEC O157, the bacterium was inoculated onto a sterilized rectangular cotton wool (width 20 × depth 40 × height 4 mm) that was placed onto PDA as ‘control with cotton wool’. After 7-day incubation at 25°C, the agar strip was crushed and suspended in PBS. The suspension was mixed at a full speed by using an automatic mixer and centrifuged at 4000 × g for 10 min. The supernatant was removed, and the pellet washed twice with PBS. The pellet was re-suspended again with PBS and was diluted 100-fold into 10 ml of EG medium acidified with hydrogen chloride at pH 2.5. EG medium is commonly used in evaluating acid resistance of E. coli (Lin et al., 1996). The broth was incubated at 37°C, and the population of STEC O157 was measured at 0, 1, 2 and 4 h. To enumerate STEC O157, the inoculated broth was serially diluted with PBS and pour-plated onto TSA. After incubation for 48 h at 37°C, colonies were counted. D values were calculated using the formula (D value = −1/slope), where slope represents the linear regression of the data including all the sampling points. The R2 values of the linear regression analyses were more than 0.8 in every analysis. The experiments were performed in triplicate. Error bars represent the standard deviation of the three trials. Each letter in the figures represents a significant difference by Student's t-test as follows:A. P < 0.05 compared to the control.B. P < 0.05 compared to the control with cotton wool.

Mentions: A biofilm-like structure of STEC O157 on fungal hyphae was observed under microscopic observation (shown as asterisks in Fig. 1). In various bacterial species, biofilm growth affected their stress resistance (Dykes et al., 2003; Kubota et al., 2009). Therefore, stress resistance of STEC O157 after co-culture with fungi was investigated using an acid resistance assay. Acid stress was chosen as STEC O157 must survive in the acidic gastric fluid for causing infections in humans. Briefly, after co-culture with fungi for 7 days at 25°C, STEC O157 was collected from the colony and inoculated into minimal E glucose medium (EG medium) (Vogel and Bonner, 1956), acidified with hydrogen chloride (Kanto Chemical Co., Inc., Tokyo, Japan) at pH 2.5. As an indicator for acid stress resistance in STEC O157, decimal reduction time (D value) was used. D value is the time required at a certain environment, such as heat and osmotic pressure, to decrease 90% of the organisms and is commonly used to explore appropriate control measures to a pathogen. (Barkley and Richardson, 1994) The larger values mean greater resistance of the bacteria used. In our study, the motile strain had greater resistance than the non-motile strain (Fig. 2). Variation in acid resistance among strains of STEC O157 has been known (Lee et al., 2012b), and these strains would have genetic difference in acid resistance. D values of both the motile and non-motile STEC O157 strains co-cultured with A. alternata, Colletotrichum sp., C. sphaerospermum, G. candidum, P. camemberti, P. nalgiovense, P. roqueforti and Rhizopus sp. were significantly higher (Student's t test; P < 0.01) than those of the control, which is monoculture of STEC O157 on PDA (Fig. 2). In contrast, D values of the motile strain of STEC O157 after co-culture with A. ochraceus and E. nidulans were significantly lower (Student's t test; P < 0.01) than that of the control. Co-culture with F. oxysporum did not affect the D value of the motile strain; however, in the case of non-motile strain of STEC O157, D values after co-culture with A. ochraceus, E. nidulans and F. oxysporum were significantly higher (Student's t test; P < 0.01).


Spread and change in stress resistance of Shiga toxin-producing Escherichia coli O157 on fungal colonies.

Lee K, Kobayashi N, Watanabe M, Sugita-Konishi Y, Tsubone H, Kumagai S, Hara-Kudo Y - Microb Biotechnol (2013)

D values at pH 2.5 of (A) motile and (B) non-motile strains of STEC O157 after co-culture with various fungi.Approximately 108 CFU of motile or non-motile STEC O157 was inoculated onto a 7-day-old fungal colony grown on PDA. As control, the same amount of STEC O157 was inoculated onto PDA without fungi. In addition, to investigate the effect of a filamentous structure per se on the stress resistance of STEC O157, the bacterium was inoculated onto a sterilized rectangular cotton wool (width 20 × depth 40 × height 4 mm) that was placed onto PDA as ‘control with cotton wool’. After 7-day incubation at 25°C, the agar strip was crushed and suspended in PBS. The suspension was mixed at a full speed by using an automatic mixer and centrifuged at 4000 × g for 10 min. The supernatant was removed, and the pellet washed twice with PBS. The pellet was re-suspended again with PBS and was diluted 100-fold into 10 ml of EG medium acidified with hydrogen chloride at pH 2.5. EG medium is commonly used in evaluating acid resistance of E. coli (Lin et al., 1996). The broth was incubated at 37°C, and the population of STEC O157 was measured at 0, 1, 2 and 4 h. To enumerate STEC O157, the inoculated broth was serially diluted with PBS and pour-plated onto TSA. After incubation for 48 h at 37°C, colonies were counted. D values were calculated using the formula (D value = −1/slope), where slope represents the linear regression of the data including all the sampling points. The R2 values of the linear regression analyses were more than 0.8 in every analysis. The experiments were performed in triplicate. Error bars represent the standard deviation of the three trials. Each letter in the figures represents a significant difference by Student's t-test as follows:A. P < 0.05 compared to the control.B. P < 0.05 compared to the control with cotton wool.
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fig02: D values at pH 2.5 of (A) motile and (B) non-motile strains of STEC O157 after co-culture with various fungi.Approximately 108 CFU of motile or non-motile STEC O157 was inoculated onto a 7-day-old fungal colony grown on PDA. As control, the same amount of STEC O157 was inoculated onto PDA without fungi. In addition, to investigate the effect of a filamentous structure per se on the stress resistance of STEC O157, the bacterium was inoculated onto a sterilized rectangular cotton wool (width 20 × depth 40 × height 4 mm) that was placed onto PDA as ‘control with cotton wool’. After 7-day incubation at 25°C, the agar strip was crushed and suspended in PBS. The suspension was mixed at a full speed by using an automatic mixer and centrifuged at 4000 × g for 10 min. The supernatant was removed, and the pellet washed twice with PBS. The pellet was re-suspended again with PBS and was diluted 100-fold into 10 ml of EG medium acidified with hydrogen chloride at pH 2.5. EG medium is commonly used in evaluating acid resistance of E. coli (Lin et al., 1996). The broth was incubated at 37°C, and the population of STEC O157 was measured at 0, 1, 2 and 4 h. To enumerate STEC O157, the inoculated broth was serially diluted with PBS and pour-plated onto TSA. After incubation for 48 h at 37°C, colonies were counted. D values were calculated using the formula (D value = −1/slope), where slope represents the linear regression of the data including all the sampling points. The R2 values of the linear regression analyses were more than 0.8 in every analysis. The experiments were performed in triplicate. Error bars represent the standard deviation of the three trials. Each letter in the figures represents a significant difference by Student's t-test as follows:A. P < 0.05 compared to the control.B. P < 0.05 compared to the control with cotton wool.
Mentions: A biofilm-like structure of STEC O157 on fungal hyphae was observed under microscopic observation (shown as asterisks in Fig. 1). In various bacterial species, biofilm growth affected their stress resistance (Dykes et al., 2003; Kubota et al., 2009). Therefore, stress resistance of STEC O157 after co-culture with fungi was investigated using an acid resistance assay. Acid stress was chosen as STEC O157 must survive in the acidic gastric fluid for causing infections in humans. Briefly, after co-culture with fungi for 7 days at 25°C, STEC O157 was collected from the colony and inoculated into minimal E glucose medium (EG medium) (Vogel and Bonner, 1956), acidified with hydrogen chloride (Kanto Chemical Co., Inc., Tokyo, Japan) at pH 2.5. As an indicator for acid stress resistance in STEC O157, decimal reduction time (D value) was used. D value is the time required at a certain environment, such as heat and osmotic pressure, to decrease 90% of the organisms and is commonly used to explore appropriate control measures to a pathogen. (Barkley and Richardson, 1994) The larger values mean greater resistance of the bacteria used. In our study, the motile strain had greater resistance than the non-motile strain (Fig. 2). Variation in acid resistance among strains of STEC O157 has been known (Lee et al., 2012b), and these strains would have genetic difference in acid resistance. D values of both the motile and non-motile STEC O157 strains co-cultured with A. alternata, Colletotrichum sp., C. sphaerospermum, G. candidum, P. camemberti, P. nalgiovense, P. roqueforti and Rhizopus sp. were significantly higher (Student's t test; P < 0.01) than those of the control, which is monoculture of STEC O157 on PDA (Fig. 2). In contrast, D values of the motile strain of STEC O157 after co-culture with A. ochraceus and E. nidulans were significantly lower (Student's t test; P < 0.01) than that of the control. Co-culture with F. oxysporum did not affect the D value of the motile strain; however, in the case of non-motile strain of STEC O157, D values after co-culture with A. ochraceus, E. nidulans and F. oxysporum were significantly higher (Student's t test; P < 0.01).

Bottom Line: To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin-producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food-related fungi including fermentation starters.The population of STEC O157 increased when co-cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus.After co-culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

Show MeSH
Related in: MedlinePlus