scholarly journals First Evidence of Division and Accumulation of Viable but Nonculturable Pseudomonas fluorescens Cells on Surfaces Subjected to Conditions Encountered at Meat Processing Premises

2007 ◽  
Vol 73 (9) ◽  
pp. 2839-2846 ◽  
Author(s):  
Sophie Peneau ◽  
Danielle Chassaing ◽  
Brigitte Carpentier
Keyword(s):  
Pseudomonas Fluorescens ◽  
Food Industry ◽  
Striking Difference ◽  
Lag Phase ◽  
Tetrazolium Chloride ◽  
Cleaning Product ◽  
Viable But Nonculturable ◽  
Nonculturable Cells ◽  
Cleaning And Disinfection ◽  
Number Of Cells

ABSTRACT Cleaning and disinfection of open surfaces in food industry premises leave some microorganisms behind; these microorganisms build up a resident flora on the surfaces. Our goal was to explore the phenomena involved in the establishment of this biofilm. Ceramic coupons were contaminated, once only, with Pseudomonas fluorescens suspended in meat exudate incubated at 10°C. The mean adhering population after 1 day was 102 CFU·cm−2 and 103 total cells·cm−2, i.e., the total number of cells stained by DAPI (4′,6′-diamidino-2-phenylindole). The coupons were subjected daily to a cleaning product, a disinfectant, and a further soiling with exudate. The result was a striking difference between the numbers of CFU, which reached 104 CFU·cm−2, and the numbers of total cells, which reached 2 × 106 cells·cm−2 in 10 days. By using hypotheses all leading to an overestimation of the number of dead cells, we showed that the quantity of nonculturable cells (DAPI-positive cells minus CFU) observed cannot be accounted for as an accumulation of dead cells. Some nonculturable cells are therefore dividing on the surface, although cell division is unable to continue to the stage of macrocolony formation on agar. The same phenomenon was observed when only a chlorinated alkaline product was used and the number of cells capable of reducing 5-cyano-2,3-ditolyl tetrazolium chloride was close to the number of total cells, confirming that most nonculturable cells are viable but nonculturable. Furthermore, the daily shock applied to the cells does not prompt them to enter a new lag phase. Since a single application of microorganisms is sufficient to produce this accumulation of cells, it appears that the phenomenon is inevitable on open surfaces in food industry premises.

SYBR green as a fluorescent probe to evaluate the biofilm physiological state of Staphylococcus epidermidis, using flow cytometry

2011 ◽  
Vol 57 (10) ◽  
pp. 850-856 ◽  
Author(s):  
Filipe Cerca ◽  
Gabriela Trigo ◽  
Alexandra Correia ◽  
Nuno Cerca ◽  
Joana Azeredo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Staphylococcus Epidermidis ◽  
Physiological State ◽  
Staining Intensity ◽  
Sybr Green ◽  
Tetrazolium Chloride ◽  
Viable But Nonculturable ◽  
Bacterial Rna ◽  
Nonculturable Cells ◽  
Excess Glucose

Staphylococcus epidermidis biofilms with different proportions of viable but nonculturable bacteria were used to show that SYBR green (SYBR) may be used as a probe to evaluate the bacterial physiological state using flow cytometry. Biofilms grown in excess glucose presented significantly higher proportions of dormant bacteria than biofilms grown in excess glucose with buffered pH conditions or with exponential-phase planktonic cultures. Bacteria obtained from biofilms with high or low proportions of viable but nonculturable cells were further cultured in broth medium and stained with SYBR at different time points. An association between bacterial growth and SYBR staining intensity was observed. In addition, bacteria presenting higher SYBR fluorescence intensity also stained more intensely with cyanoditolyl tetrazolium chloride, used as a probe to evaluate cellular metabolism. Accordingly, planktonic bacteria treated with rifampicin, an inhibitor of bacterial RNA transcription, presented lower SYBR and cyanoditolyl tetrazolium chloride staining intensity than nontreated bacteria. Overall, our results indicate that SYBR, in addition to being used as a component of LIVE/DEAD stain, may also be used as a probe to evaluate the physiological state of S. epidermidis cells.

scholarly journals Microbial Biofilms in the Food Industry—A Comprehensive Review

2021 ◽  
Vol 18 (4) ◽  
pp. 2014
Author(s):  
Conrado Carrascosa ◽  
Dele Raheem ◽  
Fernando Ramos ◽  
Ariana Saraiva ◽  
António Raposo
Keyword(s):  
Food Industry ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Microbial Biofilms ◽  
Formidable Challenge ◽  
Electrostatic Charging ◽  
Associated Proteins ◽  
Intercellular Signalling ◽  
Cleaning And Disinfection ◽  
Extracellular Polymeric Substance Matrix

Biofilms, present as microorganisms and surviving on surfaces, can increase food cross-contamination, leading to changes in the food industry’s cleaning and disinfection dynamics. Biofilm is an association of microorganisms that is irreversibly linked with a surface, contained in an extracellular polymeric substance matrix, which poses a formidable challenge for food industries. To avoid biofilms from forming, and to eliminate them from reversible attachment and irreversible stages, where attached microorganisms improve surface adhesion, a strong disinfectant is required to eliminate bacterial attachments. This review paper tackles biofilm problems from all perspectives, including biofilm-forming pathogens in the food industry, disinfectant resistance of biofilm, and identification methods. As biofilms are largely responsible for food spoilage and outbreaks, they are also considered responsible for damage to food processing equipment. Hence the need to gain good knowledge about all of the factors favouring their development or growth, such as the attachment surface, food matrix components, environmental conditions, the bacterial cells involved, and electrostatic charging of surfaces. Overall, this review study shows the real threat of biofilms in the food industry due to the resistance of disinfectants and the mechanisms developed for their survival, including the intercellular signalling system, the cyclic nucleotide second messenger, and biofilm-associated proteins.

scholarly journals Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

2006 ◽  
Vol 72 (5) ◽  
pp. 3482-3488 ◽  
Author(s):  
M�nica Ordax ◽  
Ester Marco-Noales ◽  
Mar�a M. L�pez ◽  
Elena G. Biosca
Keyword(s):  
Pathogenic Bacteria ◽  
Erwinia Amylovora ◽  
Control Plant ◽  
Viable Cell ◽  
Epifluorescence Microscopy ◽  
Survival Strategy ◽  
Vbnc State ◽  
Tetrazolium Chloride ◽  
Viable But Nonculturable ◽  
Cell Counts

ABSTRACT Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.

Antibacterial Effects of Long-Chain Polyphosphates on Selected Spoilage and Pathogenic Bacteria

2008 ◽  
Vol 71 (7) ◽  
pp. 1401-1405 ◽  
Author(s):  
JEREMY A. OBRITSCH ◽  
DOJIN RYU ◽  
LUCINA E. LAMPILA ◽  
LLOYD B. BULLERMAN
Keyword(s):  
Food Industry ◽  
Pathogenic Bacteria ◽  
Antimicrobial Activities ◽  
Lag Phase ◽  
E Coli ◽  
Inhibition Of Growth ◽  
K 12 ◽  
Chain Lengths ◽  
And Staphylococcus Aureus ◽  
Long Chain

The antimicrobial activities of four long-chain food-grade polyphosphates were studied at concentrations allowed in the food industry (<5,000 ppm) in defined basal media by determining the inhibition of growth of three gram-negative and four gram-positive spoilage and pathogenic bacteria. Both generation time and lag phase of Escherichia coli K-12, E. coli O157: H7, and Salmonella Typhimurium were increased with all of the polyphosphates tested. Bacillus subtilis and Staphylococcus aureus were more sensitive to polyphosphates, but not in all cases, with multiphased growth. The growth of Lactobacillus plantarum was inhibited by polyphosphates at concentrations above 750 ppm, but the lag time of Listeria monocytogenes was shortened by the presence of polyphosphates. No single polyphosphate was maximally inhibitory against all bacteria. Polyphosphates with chain lengths of 12 to 15 were significantly different from those with chain lengths of 18 to 21 depending on the organism and concentrations of polyphosphate used. Overall, higher polyphosphate concentrations resulted in greater inhibition of bacterial growth.

SIGNIFICANCE OF VIABLE BUT NONCULTURABLE BACTERIA FOR SAFETY OF FOOD PRODUCTS

2019 ◽  
Vol 1 (2) ◽  
pp. 183-189
Author(s):  
A. M. Abdullaeva ◽  
◽  
L. P. Blinkova ◽  
Yu. D. Pakhomov ◽  
◽  
...  
Keyword(s):  
Food Products ◽  
Active State ◽  
Insufficient Data ◽  
Main Hypothesis ◽  
Viable But Nonculturable ◽  
Nonculturable Cells

In this review data on hazardous influence of nonculturable cells of pathogens on humans and animals, of contamination of foodstuffs is presented and also attention is stressed on properties of such cells and their effect through foodstuffs on humans and animals. Main hypothesis of formation and resuscitation of viable but nonculturable cells are elucidated. Factors that influence shifting bacteria to nonculturability and their conversion into active state are discussed. The conclusion is drawn about biohazard of viable nonculturable cells and insufficient data about their physiology and mechanisms of transition into this state and resuscitation back.

scholarly journals Microbial Scout Hypothesis, Stochastic Exit from Dormancy, and the Nature of Slow Growers

2012 ◽  
Vol 78 (9) ◽  
pp. 3221-3228 ◽  
Author(s):  
S. Buerger ◽  
A. Spoering ◽  
E. Gavrish ◽  
C. Leslin ◽  
L. Ling ◽  
...  
Keyword(s):  
Central Element ◽  
Low Frequency ◽  
Adaptive Mutation ◽  
Lag Phase ◽  
Content Type ◽  
Microbial Life ◽  
Delayed Initiation ◽  
Nonculturable Cells ◽  
Visible Growth ◽  
Species Specific

ABSTRACTWe recently proposed a scout model of the microbial life cycle (S. S. Epstein, Nature 457:1083, 2009), the central element of which is the hypothesis that dormant microbial cells wake up into active (so-called scout) cells stochastically, independently of environmental cues. Here, we check the principal prediction of this hypothesis: under growth-permissive conditions, dormant cells initiate growth at random time intervals and exhibit no species-specific lag phase. We show that a range of microorganisms, including environmental species,Escherichia coli, andMycobacterium smegmatis, indeed wake up in a seemingly stochastic manner and independently of environmental conditions, even in the longest incubations conducted (months to years long). As is implicit in the model, most of the cultures we obtained after long incubations were not inherently slow growers. Of the environmental isolates that required ≥7 months to form visible growth, only 5% needed an equally long incubation upon subculturing, with the majority exhibiting regrowth within 24 to 48 h. This apparent change was not a result of adaptive mutation; rather, most microbial species that appear to be slow growers were in fact fast growers with a delayed initiation of division. Genuine slow growth thus appears to be less significant than previously believed. Random, low-frequency exit from the nongrowing state may be a key element of a general microbial survival strategy, and the phylogenetic breadth of the organisms exhibiting such exit indicates that it represents a general phenomenon. The stochasticity of awakening can also provide a parsimonious explanation to several microbiological observations, including the apparent randomness of latent infections and the existence of viable-but-nonculturable cells (VBNC).

Involvement ofgacSandrpoSin enhancement of the plant growth-promoting capabilities ofEnterobacter cloacaeCAL2 and UW4

2001 ◽  
Vol 47 (8) ◽  
pp. 698-705 ◽  
Author(s):  
Saleema S Saleh ◽  
Bernard R Glick
Keyword(s):  
Plant Growth ◽  
Pseudomonas Fluorescens ◽  
Indoleacetic Acid ◽  
Acc Deaminase ◽  
Enterobacter Cloacae ◽  
Lag Phase ◽  
Plant Growth Promoting Bacteria ◽  
Multicopy Plasmid ◽  
Plant Growth Promoting ◽  
Growth Promoting

The plant growth-promoting bacteria Enterobacter cloacae CAL2 and UW4 were genetically transformed with a multicopy plasmid containing an rpoS or gacS gene from Pseudomonas fluorescens. The transformed strains were compared with the nontransformed strains for growth, indoleacetic acid (IAA) production, antibiotic production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, siderophore production, cell morphology, and the ability to promote canola root elongation. All transformed strains had a longer lag phase, were slower in reaching stationary phase, and attained a higher cell density than the nontransformed strains. Transformation resulted in cells that were significantly shorter than the nontransformed cells. The transformed strains also produced significantly more IAA than the nontransformed strains. Introduction of rpoS or gacS from Pseudomonas fluorescens was associated with a reduction in the production of both antibiotics, 2,4-diacetylphloroglucinol and mono-acetylphloroglucinol, produced by Enterobacter cloacae CAL2. With Enterobacter cloacae CAL2, plasmid-borne rpoS, but not gacS, increased the level of ACC deaminase activity, while introduction of rpoS in Enterobacter cloacae UW4 caused a decrease in ACC deaminase activity. Neither gacS nor rpoS significantly affected the level of siderophores synthesized by either bacterial strain. Overproduction of either GacA or RpoS in Enterobacter cloacae CAL2 resulted in a significant increase in the root lengths of canola seedlings when seeds were treated with the bacteria, and overproduction of RpoS caused an increase in canola shoot as well as root lengths.Key words: plant growth-promoting bacteria, canola, ethylene, ACC deaminase, GacS, RpoS, indoleacetic acid, siderophores, antibiotics.

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