Bacteriophages Infecting Gram-Negative Bacteria in a Commercial Cucumber Fermentation

Zhongjing Lu,Fred Breidt,Ilenys M Pérez-Díaz,Janet S Hayes

doi:10.3389/fmicb.2020.01306

Abstract

Cucumber fermentations are one of the most important vegetable fermentations in the United States. The fermentation is usually driven by lactic acid bacteria (LAB) indigenous to fresh cucumbers. But LAB are greatly outnumbered by many Gram-negative bacteria on fresh cucumbers, which may influence the growth of LAB and the incidence of bloater defect (hollow cavities formed inside fermented cucumbers) leading to serious economic loss to the pickle industry. Rapid elimination of Gram-negative bacteria is crucial to the dominance of LAB and the reduction of bloater defect in the fermentation. Various factors can affect the viability of Gram-negative bacteria in cucumber fermentation. Bacteriophages (phages) may be one of such factors. This study explored the abundance, diversity, and functional role of phages infecting Gram-negative bacteria in a commercial cucumber fermentation. Cover brine samples were taken from a commercial fermentation tank over a 30-day period. On day 1 and day 3 of the fermentation, 39 Gram-negative bacteria and 26 independent phages were isolated. Nearly 67% of Gram-negative bacterial isolates were susceptible to phage infection. Phage hosts include Enterobacter, Citrobacter, Escherichia, Pantoea, Serratia, Leclercia, Providencia, and Pseudomonas species. About 88% of the isolated phages infected the members in the family Enterobacteriaceae and 58% of phages infected Enterobacter species. Eight phages with unique host ranges were characterized. These phages belong to the Myoviridae, Siphoviridae, or Podoviridae family and showed distinct protein profiles and DNA fingerprints. The infectivity of a phage against Enterobacter cancerogenus was evaluated in cucumber juice as a model system. The phage infection at the multiplicity of infection 1 or 100 resulted in a 5-log reduction in cell concentration within 3 h and rapidly eliminated its host. This study revealed the abundance and variety of phages infecting Gram-negative bacteria, particularly Enterobacteriaceae, in the commercial cucumber fermentation, suggesting that phages may play an important role in the elimination of Gram-negative bacteria, thereby facilitating the dominance of LAB and minimizing bloater defect. To our knowledge, this is the first report on the ecology of phages infecting Gram-negative bacteria in commercial cucumber fermentations.

Highlights

Vegetable fermentation plays an important role in preserving foods and providing variety to the diet as well as enhancing the nutrient content of the food
The results showed that the infection of E. cancerogenus 107 by its phage 107 at the multiplicity of infection (MOI) of 1 or 100 effectively eliminated the host in cucumber juice within a short period of time in the absence of lactic acid bacteria (LAB) and high salt concentration, the two factors thought to be critical in the die off of Gram-negative bacteria in cucumber fermentations
The data from this study showed that abundant and diverse phages were present in a commercial cucumber fermentation, and 67% of Gram-negative bacterial isolates were sensitive to phage infection

Summary

Introduction

Vegetable fermentation plays an important role in preserving foods and providing variety to the diet as well as enhancing the nutrient content of the food. An average production of pickles in the United States is around 480,000 tons in 2012–2014 with an economic value between 145 and 175 million dollars (Anonymous, 2015). Gram-negative bacteria are typically predominant on fresh cucumbers (Pederson and Albury, 1950; Mundt et al, 1967; Mundt and Hammer, 1968; Mundt, 1970; Pérez-Díaz et al, 2019). The dominance of Gram-negative bacteria can interfere with the growth of LAB resulting in slow fermentation and affect thequality and/or safety of fermented cucumbers. Pathogenic bacteria have not been reported in commercially fermented vegetable products, the survival of certain acid-resistant bacterial pathogens can occur (Breidt and Caldwell, 2011). The key to ensure successful cucumber fermentation as well as other vegetable fermentations is to establish conditions to promote the growth of LAB over other microorganisms, Gram-negative bacteria so that LAB can quickly dominate in the fermentation system before Gram-negative bacteria and other undesirable microorganisms have a chance to increase substantially in numbers and exert their effects

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