Detection and Verification of the Viable but Nonculturable (VBNC) State of <i>Escherichia coli</i> O157:H7 and <i>Listeria monocytogenes</i> Using Flow Cytometry and Standard Plating

Afari, George Kwabena; Hung, Yen‐Con

doi:10.1111/1750-3841.14203

Cited by 36 publications

(25 citation statements)

References 38 publications

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“…Thus, VBNC cell count can be estimated by subtracting the number of culturable cells from the total viable cell count determined using PMA-qPCR. This technique has been used for the detection of different VBNC bacterial cells, such as Escherichia coli and Vibrio parahaemolyticus (Afari and Hung, 2018;Chang and Lin, 2018;Zhong and Zhao, 2018;Telli and Dogruer, 2019).…”

Section: Introductionmentioning

confidence: 99%

Detection and Quantification of Viable but Non-culturable Campylobacter jejuni

Wang

Feng

et al. 2020

Front. Microbiol.

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Campylobacter can enter a viable but non-culturable (VBNC) state to evade various stresses, and this state is undetectable using traditional microbiological culturing techniques. These VBNC bacterial cells retain metabolism and demonstrate pathogenic potential due to their ability to resuscitate under favorable conditions. Rapid and accurate determination of VBNC Campylobacter is critical to further understand the induction and resuscitation of the dormancy state of this microbe in the agri-food system. Here, we integrated propidium monoazide (PMA) with real-time polymerase chain reaction (qPCR) targeting the rpoB gene to detect and quantify Campylobacter jejuni in the VBNC state. First, we optimized the concentration of PMA (20 µM) that could significantly inhibit the amplification of dead cells by qPCR with no significant interference on the amplification of viable cell DNA. PMA-qPCR was highly specific to C. jejuni with a limit of detection (LOD) of 2.43 log CFU/ml in pure bacterial culture. A standard curve for C. jejuni cell concentrations was established with the correlation coefficient of 0.9999 at the linear range of 3.43 to 8.43 log CFU/ml. Induction of C. jejuni into the VBNC state by osmotic stress (i.e., 7% NaCl) was rapid (<48 h) and effective (>10% population). The LOD of PMA-qPCR for VBNC C. jejuni exogenously applied to chicken breasts was 3.12 log CFU/g. In conclusion, PMA-qPCR is a rapid, specific, and sensitive method for the detection and quantification of VBNC C. jejuni in poultry products. This technique can give insight into the prevalence of VBNC Campylobacter in the environment and agri-food production system.

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Section: Introductionmentioning

confidence: 99%

Detection and Quantification of Viable but Non-culturable Campylobacter jejuni

Wang

Feng

et al. 2020

Front. Microbiol.

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“…Early VBNC studies coupled bacterial culture with microscopic counting Microorganisms 2020, 8, 184; doi:10.3390/microorganisms8020184 www.mdpi.com/journal/microorganisms of stained cells using SYTO 9/propidium iodide (SYTO9/PI), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), 4 ,6-diamidino-2-phenylindole (DAPI), and/or fluorescently labelled antibodies [7,[10][11][12]. Recent studies expanded the methodological approaches to determine the metabolic activity and cell membrane integrity of VBNC cells by ATP determination [13], quantitative PCR combined with propidium monoazide treatment [14], the capability to ferment sugars after reuptake [13], and flow cytometric analyses coupled with cell staining [15]. Flow cytometric analysis of SYTO9/PI was also linked to the uptake activity of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to assess both cell membrane integrity and metabolic activity simultaneously [16], thereby quantifying VBNC bacteria at the single cell level [17].…”

Section: Introductionmentioning

confidence: 99%

“…Flow cytometric analysis of SYTO9/PI was also linked to the uptake activity of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to assess both cell membrane integrity and metabolic activity simultaneously [16], thereby quantifying VBNC bacteria at the single cell level [17]. Various stress conditions have been identified to induce the VBNC state in L. monocytogenes, including chlorine-based sanitizing treatments [15] and non-ionic surfactants combined with inorganic salts [13]. Quaternary ammonium compounds (QACs), such as benzalkonium chloride (BC), are widely used as biocides for the disinfection of surfaces, including food production environments [18,19].…”

Section: Introductionmentioning

confidence: 99%

Benzalkonium Chloride Induces a VBNC State in Listeria monocytogenes

et al. 2020

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The objective of our study was to investigate the effects of benzalkonium chloride (BC) adaptation of L. monocytogenes on the susceptibility to antimicrobial agents and on the viable but non culturable (VBNC) state of the bacterial cells. We adapted L. monocytogenes SLCC2540 to BC by applying BC below minimum inhibitory concentration (MIC) to above minimum bactericidal concentration (MBC). The culturable fractions and the susceptibility of adapted and parental cells to BC were assessed. In addition, cell membrane permeability and glucose uptake were analyzed by multi parametric flow cytometry using the fluorescent agents SYTO9, propidium iodide, and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG). Adapted cells displayed a two-fold MIC increase of BC and reduced antibiotic susceptibility. At high BC concentrations, the decrease in the number of colony forming units was significantly lower in the population of adapted cells compared to parental cells. At the same time, the number of metabolically active cells with intact membranes was significantly higher than the number of culturable cells. Growth-independent viability assays revealed an adapted subpopulation after BC application that was not culturable, indicating increased abundance of viable but nonculturable (VBNC) cells. Moreover, adapted cells can outcompete non-adapted cells under sublethal concentrations of disinfectants, which may lead to novel public health risks.

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“…The availability of energy sources can improve the colony formation thus the ability to be cultured. Addition of Tween 20 (3%) has been reported to resuscitate VBNC cells of Escherichia coli O157: H7 (Afari and Hung 2018) and Salmonella enterica serovar Typhi (Zeng et al, 2013).…”

Section: Resuscitation Of Stressed or Vbnc C Sakazakiimentioning

confidence: 99%

Cronobacter sakazakii local isolates response to acid stress and their resuscitability

Hati¹,

Dewanti‐Hariyadi²,

Nuraida³

2019

Food Res.

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Cronobacter spp. has been reported to cause meningitis, necrotizing enterocolitis, and septicemia in a group of infants through the consumption of powder infant formula. These bacteria are reported to withstand various stress conditions such as heating, drying, low water activity, low pH, etc. A local isolate of Cronobacter sakazakii YRt2a was reportedly survived and entered Viable But Non-Culturable (VBNC) conditions during desiccation stress. This study aims to study the behavior of local isolates of Cronobacter spp. in response to acid stress and its resuscitability. C. sakazakii E2 and YRt2a were grown in TSB at pH 3.0±0.2 or 3.5±0.2. The number of culturable cells and viable cells were enumerated by the Total Plate Count and Direct Viable Count methods, respectively. Resuscitation was done by growing the stress or VBNC cells in TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer. The results showed that C. sakazakii E2 and YRt2a entered VBNC state after 60 mins of exposure to pH 3.0±0.2, while remained culturable after 120 minutes exposure to pH 3.5±0.2. TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer could resuscitate the stress or VBNC cells of C. sakazakii. Stress or VBNC state experienced by C. sakazakii in response to acid tends to be transient and can be resuscitated. C. sakazakii experiencing stress or VBNC may pose a risk for food safety.

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Detection and Verification of the Viable but Nonculturable (VBNC) State of Escherichia coli O157:H7 and Listeria monocytogenes Using Flow Cytometry and Standard Plating

Abstract: VBNC induction conditions for foodborne pathogens during chlorine washing treatment were determined in a broth system and the information can serve as a basis for future studies that address the prevention of VBNC formation during produce wash treatments.

Cited by 36 publications

References 38 publications

Detection and Quantification of Viable but Non-culturable Campylobacter jejuni

Detection and Quantification of Viable but Non-culturable Campylobacter jejuni

Benzalkonium Chloride Induces a VBNC State in Listeria monocytogenes

Cronobacter sakazakii local isolates response to acid stress and their resuscitability

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