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Nitrogen gas flushing can be bactericidal: the temperature-dependent destiny of Bacillus weihenstephanensis KBAB4 under a pure N2 atmosphere.

Munsch-Alatossava P, Alatossava T - Front Microbiol (2014)

Bottom Line: Bacillus weihenstephanensis, a frequent inhabitant of fluid dairy products, is represented by the genome-sequenced KBAB4 strain.Among Pseudomonas, P. tolaasii LMG 2342(T) and strain C1, a raw milk psychrotroph, were selected.This observation constitutes the first evidence that N2 gas flushing has bactericidal effects.

View Article: PubMed Central - PubMed

Affiliation: Division of Food Technology, Department of Food and Environmental Sciences, University of Helsinki Helsinki, Finland.

ABSTRACT
Gram-negative Pseudomonas and Gram-positive Bacillus are the most common spoilage bacteria in raw and pasteurized milk, respectively. In previous studies, nitrogen (N2) gas flushing treatments of raw and pasteurized milk at cold chain-temperatures inhibited bacterial spoilage and highlighted different susceptibilities to the N2 treatment with the exclusion of certain bacterial types. Here, we investigated the effects of pure N2 gas flushing on representative strains of these genera grown in mono- or co-cultures at 15 and 25°C. Bacillus weihenstephanensis, a frequent inhabitant of fluid dairy products, is represented by the genome-sequenced KBAB4 strain. Among Pseudomonas, P. tolaasii LMG 2342(T) and strain C1, a raw milk psychrotroph, were selected. The N2 gas flushing treatment revealed: (1) temperature-dependent responses; (2) inhibition of the growth of both pseudomonads; (3) emergence of small colony variants (SCVs) for B. weihenstephanensis strain KBAB4 at 15°C induced by the N2 treatment or when grown in co-culture with Pseudomonas strains; (4) N2 gas flushing modulates (suppressed or stimulated) bacterial antagonistic reactions in co-cultures; (5) most importantly, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed that at 25°C the majority of the KBAB4 cells were killed by pure N2 gas flushing. This observation constitutes the first evidence that N2 gas flushing has bactericidal effects.

No MeSH data available.


Related in: MedlinePlus

Time-course analyses of the number (cfu/ml) of B. weihenstephanensis KBAB4 cells (K) established as mono- or co-cultures in BHI broth at 25°C with (A) a Pseudomonas raw milk isolate C1 (C1) or (B) P. tolaasii LMG 2342T (42). Similarly, the number (cfu/ml) of (C)Pseudomonas strain C1 (C1) or (D) LMG 2342T (42) cells as mono- or co-cultures with Bacillus strain KBAB4 under continuous pure N2 gas flushing (N) together with the control conditions (C). Error bars indicate standard deviations. (A,B) KC, monoculture of B. weihenstephanensis KBAB4 as control; KN, monoculture of B. weihenstephanensis KBAB4 flushed with N2; K(C1)C, KBAB4 grown in the presence of C1 as control; K(C1)N, KBAB4 grown in the presence of C1 flushed with N2; K(42)C, KBAB4 grown in the presence of LMG 2342T as control; K(42)N, KBAB4 grown in the presence of LMG 2342T flushed with N2. (C) C1C, monoculture of the raw milk isolate C1 as control; C1N, monoculture of the raw milk isolate C1 flushed with N2; C1(K)C, C1 grown in the presence of KBAB4 as control; C1(K)N, C1 grown in the presence of KBAB4 flushed with N2. (D) 42C, monoculture of LMG 2342T as control; 42N, monoculture of LMG 2342T flushed with N2; 42(K)C, LMG 2342T grown in the presence of KBAB4 as control; 42(K)N, LMG 2342T grown in the presence of KBAB4 flushed with N2.
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Figure 1: Time-course analyses of the number (cfu/ml) of B. weihenstephanensis KBAB4 cells (K) established as mono- or co-cultures in BHI broth at 25°C with (A) a Pseudomonas raw milk isolate C1 (C1) or (B) P. tolaasii LMG 2342T (42). Similarly, the number (cfu/ml) of (C)Pseudomonas strain C1 (C1) or (D) LMG 2342T (42) cells as mono- or co-cultures with Bacillus strain KBAB4 under continuous pure N2 gas flushing (N) together with the control conditions (C). Error bars indicate standard deviations. (A,B) KC, monoculture of B. weihenstephanensis KBAB4 as control; KN, monoculture of B. weihenstephanensis KBAB4 flushed with N2; K(C1)C, KBAB4 grown in the presence of C1 as control; K(C1)N, KBAB4 grown in the presence of C1 flushed with N2; K(42)C, KBAB4 grown in the presence of LMG 2342T as control; K(42)N, KBAB4 grown in the presence of LMG 2342T flushed with N2. (C) C1C, monoculture of the raw milk isolate C1 as control; C1N, monoculture of the raw milk isolate C1 flushed with N2; C1(K)C, C1 grown in the presence of KBAB4 as control; C1(K)N, C1 grown in the presence of KBAB4 flushed with N2. (D) 42C, monoculture of LMG 2342T as control; 42N, monoculture of LMG 2342T flushed with N2; 42(K)C, LMG 2342T grown in the presence of KBAB4 as control; 42(K)N, LMG 2342T grown in the presence of KBAB4 flushed with N2.

Mentions: The enumeration of viable cells from KBAB4 grown in monoculture revealed that, irrespective of the conditions, both the control (KC) and the treated (KN) cultures followed a similar growth trend during the first 24 h, marking the end of the exponential growth phase. In the subsequent stationary phase, the control culminated at approximately 8.5 log-units during the remaining 5–6 days (Figures 1A,B). In contrast, under the N2 treatment, KBAB4 (KN) gradually decreased after 24 h; the level of the surviving cells was reduced by four log-units (approximately 10,000-fold) compared to the control (KC), after 5 or 6 days of flushing at 25°C (Figures 1A,B).


Nitrogen gas flushing can be bactericidal: the temperature-dependent destiny of Bacillus weihenstephanensis KBAB4 under a pure N2 atmosphere.

Munsch-Alatossava P, Alatossava T - Front Microbiol (2014)

Time-course analyses of the number (cfu/ml) of B. weihenstephanensis KBAB4 cells (K) established as mono- or co-cultures in BHI broth at 25°C with (A) a Pseudomonas raw milk isolate C1 (C1) or (B) P. tolaasii LMG 2342T (42). Similarly, the number (cfu/ml) of (C)Pseudomonas strain C1 (C1) or (D) LMG 2342T (42) cells as mono- or co-cultures with Bacillus strain KBAB4 under continuous pure N2 gas flushing (N) together with the control conditions (C). Error bars indicate standard deviations. (A,B) KC, monoculture of B. weihenstephanensis KBAB4 as control; KN, monoculture of B. weihenstephanensis KBAB4 flushed with N2; K(C1)C, KBAB4 grown in the presence of C1 as control; K(C1)N, KBAB4 grown in the presence of C1 flushed with N2; K(42)C, KBAB4 grown in the presence of LMG 2342T as control; K(42)N, KBAB4 grown in the presence of LMG 2342T flushed with N2. (C) C1C, monoculture of the raw milk isolate C1 as control; C1N, monoculture of the raw milk isolate C1 flushed with N2; C1(K)C, C1 grown in the presence of KBAB4 as control; C1(K)N, C1 grown in the presence of KBAB4 flushed with N2. (D) 42C, monoculture of LMG 2342T as control; 42N, monoculture of LMG 2342T flushed with N2; 42(K)C, LMG 2342T grown in the presence of KBAB4 as control; 42(K)N, LMG 2342T grown in the presence of KBAB4 flushed with N2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Time-course analyses of the number (cfu/ml) of B. weihenstephanensis KBAB4 cells (K) established as mono- or co-cultures in BHI broth at 25°C with (A) a Pseudomonas raw milk isolate C1 (C1) or (B) P. tolaasii LMG 2342T (42). Similarly, the number (cfu/ml) of (C)Pseudomonas strain C1 (C1) or (D) LMG 2342T (42) cells as mono- or co-cultures with Bacillus strain KBAB4 under continuous pure N2 gas flushing (N) together with the control conditions (C). Error bars indicate standard deviations. (A,B) KC, monoculture of B. weihenstephanensis KBAB4 as control; KN, monoculture of B. weihenstephanensis KBAB4 flushed with N2; K(C1)C, KBAB4 grown in the presence of C1 as control; K(C1)N, KBAB4 grown in the presence of C1 flushed with N2; K(42)C, KBAB4 grown in the presence of LMG 2342T as control; K(42)N, KBAB4 grown in the presence of LMG 2342T flushed with N2. (C) C1C, monoculture of the raw milk isolate C1 as control; C1N, monoculture of the raw milk isolate C1 flushed with N2; C1(K)C, C1 grown in the presence of KBAB4 as control; C1(K)N, C1 grown in the presence of KBAB4 flushed with N2. (D) 42C, monoculture of LMG 2342T as control; 42N, monoculture of LMG 2342T flushed with N2; 42(K)C, LMG 2342T grown in the presence of KBAB4 as control; 42(K)N, LMG 2342T grown in the presence of KBAB4 flushed with N2.
Mentions: The enumeration of viable cells from KBAB4 grown in monoculture revealed that, irrespective of the conditions, both the control (KC) and the treated (KN) cultures followed a similar growth trend during the first 24 h, marking the end of the exponential growth phase. In the subsequent stationary phase, the control culminated at approximately 8.5 log-units during the remaining 5–6 days (Figures 1A,B). In contrast, under the N2 treatment, KBAB4 (KN) gradually decreased after 24 h; the level of the surviving cells was reduced by four log-units (approximately 10,000-fold) compared to the control (KC), after 5 or 6 days of flushing at 25°C (Figures 1A,B).

Bottom Line: Bacillus weihenstephanensis, a frequent inhabitant of fluid dairy products, is represented by the genome-sequenced KBAB4 strain.Among Pseudomonas, P. tolaasii LMG 2342(T) and strain C1, a raw milk psychrotroph, were selected.This observation constitutes the first evidence that N2 gas flushing has bactericidal effects.

View Article: PubMed Central - PubMed

Affiliation: Division of Food Technology, Department of Food and Environmental Sciences, University of Helsinki Helsinki, Finland.

ABSTRACT
Gram-negative Pseudomonas and Gram-positive Bacillus are the most common spoilage bacteria in raw and pasteurized milk, respectively. In previous studies, nitrogen (N2) gas flushing treatments of raw and pasteurized milk at cold chain-temperatures inhibited bacterial spoilage and highlighted different susceptibilities to the N2 treatment with the exclusion of certain bacterial types. Here, we investigated the effects of pure N2 gas flushing on representative strains of these genera grown in mono- or co-cultures at 15 and 25°C. Bacillus weihenstephanensis, a frequent inhabitant of fluid dairy products, is represented by the genome-sequenced KBAB4 strain. Among Pseudomonas, P. tolaasii LMG 2342(T) and strain C1, a raw milk psychrotroph, were selected. The N2 gas flushing treatment revealed: (1) temperature-dependent responses; (2) inhibition of the growth of both pseudomonads; (3) emergence of small colony variants (SCVs) for B. weihenstephanensis strain KBAB4 at 15°C induced by the N2 treatment or when grown in co-culture with Pseudomonas strains; (4) N2 gas flushing modulates (suppressed or stimulated) bacterial antagonistic reactions in co-cultures; (5) most importantly, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed that at 25°C the majority of the KBAB4 cells were killed by pure N2 gas flushing. This observation constitutes the first evidence that N2 gas flushing has bactericidal effects.

No MeSH data available.


Related in: MedlinePlus