Salmonella enterica Filamentation Induced by Pelargonic Acid Is a Transient Morphotype

Abstract

Under stressful conditions, Salmonella enterica forms multinucleated elongated filaments. The triggers and outcomes of filamentation are not well characterized. S. enterica serotypes Newport, Javiana, and Typhimurium were evaluated for their ability to form filaments upon exposure to 20 mM pelargonic acid. S. Newport was used as a model to investigate the progression and fate of filamentation via culturable population size, cell length, and viability assays. All serotypes displayed filament formation after 16 h of incubation. Pelargonic acid amendment of tryptic soy broth (TSBpel) produced a 5-log CFU reduction compared to TSB after 24 h (P < 0.05), and the growth rate decreased (P < 0.02). Cell elongation started within 12 h, peaked at 16 h, and was followed by filament disintegration at 20 to 24 h. The ratio of filaments to regular-sized cells (F/R) in TSBpel was 3.87 ± 0.59 at 16 h, decreasing to 0.23 ± 0.04 and 0.03 ± 0.01 (P < 0.05) at 20 and 24 h, respectively. Mg²⁺ supplementation repressed filamentation (F/R = 0.25 ± 0.11) and enhanced culturable cell counts (P < 0.05). Continued exposure to pelargonic acid inhibited growth in TSB and M9 compared to that in unamended media (P < 0.05). However, in M9 medium without Mg²⁺ amended with 20 mM pelargonic acid (M9pel), filament fragmentation progressed independently of pelargonic acid or Mg²⁺ When cells were pretreated with pelargonic acid to induce filamentation and then transferred to fresh medium, a positive effect of Mg²⁺ was noted under nutrient-deficient conditions, with higher live/dead cell ratios in M9 supplemented with 5 mM Mg²⁺ (M9Mg) than in M9 (P < 0.05). No change was observed when pelargonic acid was also added. Filamentation was ubiquitous in all serotypes tested, transient, and sensitive to Mg²⁺ Fragmentation, but not recovery, progressed irrespective of antimicrobial or Mg²⁺ presence.IMPORTANCE Some bacteria form elongated multinucleated structures, or filaments, when exposed to stress. The filamentous form of foodborne bacterial pathogens can interfere with food protection practices and diagnostic testing. Filamentation in Salmonella enterica Newport was investigated in response to pelargonic acid, a compound naturally found in several fruit and vegetables, and also used commercially as an herbicide. Salmonella readily formed filaments when exposed to pelargonic acid. Filaments were not stable, however, and fragmented to individual cells even when the fatty acid was still present, recovering fully when the stress was alleviated. A deeper exploration of the molecular mechanisms regulating filamentation and the conditions that induce it in agriculture and the food supply chain is needed to devise strategies that curb this response.

Keywords: PhoPQ; Salmonella Newport; Salmonella filamentation; Salmonella stress mitigation; magnesium; pelargonic acid.

FIG 1

(A) Growth of Salmonella Newport cells (log CFU per milliliter) (A) over a period of 24 h in TSB, TSB with 20 mM pelargonic acid (TSBpel), TSB with 5 mM Mg²⁺ (TSBMg), and TSB with 20 mM pelargonic acid and 5 mM Mg²⁺ (TSBpelMg). (B) Fate of S. Newport cells preexposed to TSBpel for 16 h to form filaments, on transfer to fresh TSB and TSBpel. Plate counts were performed at 4 and 8 h following transfer, to determine the log CFU/ml. Error bars indicate standard error.

FIG 2

Dark-field microscopic view of Salmonella Newport in TSB amended with 20 mM pelargonic acid (TSBpel) after 12, 16, 20, and 24 h of incubation (A) and filaments of S. Newport displaying septation at 20 h and 24 h of growth in TSBpel (B). The scale bars in the bottom right corners represent 10 μm in length. (C) Schematic representation of the stages of S. Newport filamentation formed in the presence of pelargonic acid.

FIG 3

Dark-field microscopic view of Salmonella Newport following 16 h of incubation in TSB (left), TSB amended with 20 mM pelargonic acid (TSBpel) (middle), and TSB amended with 20 mM pelargonic acid and 5 mM Mg²⁺ (TSBpelMg) (right). The scale bar in the bottom right corner represents 10 μm in length.

FIG 4

Dark-field microscopic view of Salmonella Newport cells (pretreated with TSB plus 20 mM pelargonic acid for 16 h) at 0, 4, and 8 h (columns 1 to 3, respectively) in M9 minimal salts medium (row 1), M9 minimal salts medium with 20 mM pelargonic acid (M9pel, row 2), M9 minimal salts medium with 5 mM Mg²⁺ (M9Mg, row 3), and M9 minimal salts medium with 20 mM pelargonic acid and 5 mM Mg²⁺ (M9pelMg, row 4). The scale bars in the bottom right corners represent 10 μm in length.

FIG 5

Measurements of plate counts (log CFU per milliliter) (A), OD₆₀₀ (B), and live/dead ratio (C) of Salmonella Newport cells pretreated for 16 h in TSB with 20 mM pelargonic acid following transfer to M9 minimal salts medium (M9), M9 minimal salts medium with 5 mM Mg²⁺ (M9Mg), M9 minimal salts medium with 20 mM pelargonic acid (M9pel), and M9 minimal salts medium with 20 mM pelargonic acid and 5 mM Mg²⁺ (M9pelMg). Error bars indicate standard error.

FIG 6

Confocal microscopic view of Salmonella Newport (first row), S. Javiana (middle row), and S. Typhimurium (bottom row) after 16 h (left column) and 24 h (right column) exposure to TSB with 20 mM pelargonic acid. Scale bars represent 10 μm in length.