scholarly journals Induction of Viable but Nonculturable Escherichia coli O157:H7 in the Phyllosphere of Lettuce: a Food Safety Risk Factor

2011 ◽  
Vol 77 (23) ◽  
pp. 8295-8302 ◽  
Author(s):  
Laura-Dorina Dinu ◽  
Susan Bach
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Low Temperature ◽  
Cell Density ◽  
Prolonged Storage ◽  
Potential Hazard ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
E Coli

ABSTRACTEscherichia coliO157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate ofE. coliO157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction ofE. coliO157:H7 Tn7gfptransformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), bothE. coliO157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (109and 106E. coliO157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log10cells but did not detect culturable cells. These findings indicate thatE. coliO157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.

scholarly journals Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Lu Han ◽  
Kaidi Wang ◽  
Lina Ma ◽  
Pascal Delaquis ◽  
Susan Bach ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pure Culture ◽  
Fresh Produce ◽  
Isothermal Amplification ◽  
Propidium Monoazide ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
E Coli ◽  
Detection And Quantification

ABSTRACT Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the “viable but nonculturable” (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

scholarly journals Draft Genomic Sequences of Nine Extraintestinal Pathogenic Escherichia coli Isolates from Retail Chicken Skin

2018 ◽  
Vol 7 (7) ◽  
Author(s):  
Aixia Xu ◽  
Shannon Tilman ◽  
Kristy Wisser-Parker ◽  
O. Joseph Scullen ◽  
Christopher H. Sommers
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Genomic Sequences ◽  
Content Type ◽  
E Coli ◽  
Safety Research ◽  
Pathogenic Escherichia Coli ◽  
Chicken Skin

Extraintestinal pathogenic Escherichia coli strains were isolated from retail chicken skin. Here, we report the draft genomic sequences for these nine E. coli isolates, which are currently being used in agricultural and food safety research.

scholarly journals Plant-Pathogenic Oomycetes, Escherichia coli Strains, and Salmonella spp. Frequently Found in Surface Water Used for Irrigation of Fruit and Vegetable Crops in New York State

2014 ◽  
Vol 80 (16) ◽  
pp. 4814-4820 ◽  
Author(s):  
Lisa A. Jones ◽  
Randy W. Worobo ◽  
Christine D. Smart
Keyword(s):  
Escherichia Coli ◽  
Water Quality ◽  
New York ◽  
Food Safety ◽  
Surface Water ◽  
New York State ◽  
Quality Parameters ◽  
Content Type ◽  
York State ◽  
E Coli

ABSTRACTIn the United States, surface water is commonly used to irrigate a variety of produce crops and can harbor pathogens responsible for food-borne illnesses and plant diseases. Understanding when pathogens infest water sources is valuable information for produce growers to improve the food safety and production of these crops. In this study, prevalence data along with regression tree analyses were used to correlate water quality parameters (pH, temperature, turbidity), irrigation site properties (source, the presence of livestock or fowl nearby), and precipitation data to the presence and concentrations ofEscherichia coli,Salmonellaspp., and hymexazol-insensitive (HIS) oomycetes (PhytophthoraandPythiumspp.) in New York State surface waters. A total of 123 samples from 18 sites across New York State were tested forE. coliandSalmonellaspp., of which 33% and 43% were positive, respectively. Additionally, 210 samples from 38 sites were tested for HIS oomycetes, and 88% were found to be positive, with 10 species ofPhytophthoraand 11 species ofPythiumbeing identified from the samples. Regression analysis found no strong correlations between water quality parameters, site factors, or precipitation to the presence or concentration ofE. coliin irrigation sources. ForSalmonella, precipitation (≤0.64 cm) 3 days before sampling was correlated to both presence and the highest counts. Analyses for oomycetes found creeks to have higher average counts than ponds, and higher turbidity levels were associated with higher oomycete counts. Overall, information gathered from this study can be used to better understand the food safety and plant pathogen risks of using surface water for irrigation.

scholarly journals New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO 2

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Feng Zhao ◽  
Yongtao Wang ◽  
Haoran An ◽  
Yanling Hao ◽  
Xiaosong Hu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Pressure ◽  
Cell Division ◽  
State Formation ◽  
Metabolic Activity ◽  
Rna Seq ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
E Coli ◽  
State Entry

ABSTRACT The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO 2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l -serine and l -threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO 2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO 2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation. IMPORTANCE Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO 2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment.

scholarly journals Transcriptomic Analysis of Viable but Non-Culturable Escherichia coli O157:H7 Formation Induced by Low Temperature

2019 ◽  
Vol 7 (12) ◽  
pp. 634 ◽  
Author(s):  
Junliang Zhong ◽  
Xihong Zhao
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Differentially Expressed Genes ◽  
Pathogenic Bacteria ◽  
Differentially Expressed ◽  
Transmembrane Transport ◽  
Pathway Enrichment Analysis ◽  
Escherichia Coli O157 ◽  
Vbnc State ◽  
E Coli

Escherichia coli O157:H7 is one of the most common pathogenic bacteria that pose a threat to food safety. The aim of this study was to investigate the mechanisms of the formation of viable but non-culturable (VBNC) E. coli O157:H7 induced by low temperature (−20 °C) using RNA sequencing (RNA-Seq) transcriptomics analysis. The results of the present investigation revealed the presence of 2298 differentially expressed genes in VBNC cells, accounting for 46.03% of the total number of genes. Additionally, GO function and KEGG pathway enrichment analysis were performed to investigate the functional and related metabolic pathways of the differentially expressed genes. We found that the ion transport, protein synthesis, and protein transmembrane transport activities were significantly improved in the VBNC cells, indicating that E. coli O157:H7 cells synthesized a considerable amount of protein to maintain the levels of their functional metabolic processes and life activities in the VBNC state. In conclusion, we suggest that the increased synthesis of proteins such as SecY, FtsY, and Ffh might indicate that they are the key proteins involved in the improvement of the transmembrane transport activities in VBNC E. coli O157:H7 cells, maintaining their functional metabolism in the VBNC state and enhancing their survival ability under low temperatures.

scholarly journals Longitudinal Assessment of the Dynamics of Escherichia coli, Total Coliforms, Enterococcus spp., and Aeromonas spp. in Alternative Irrigation Water Sources: a CONSERVE Study

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Sultana Solaiman ◽  
Sarah M. Allard ◽  
Mary Theresa Callahan ◽  
Chengsheng Jiang ◽  
Eric Handy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Irrigation Water ◽  
Reclaimed Water ◽  
The United States ◽  
Water Sources ◽  
Agricultural Water ◽  
Content Type ◽  
E Coli ◽  
Alternative Irrigation

ABSTRACT As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (Escherichia coli, total coliforms [TC], Enterococcus, and Aeromonas). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC, enterococci (∼86%, 98%, and 90% positive, respectively; n = 333), and Aeromonas (∼98% positive; n = 133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (P < 0.001 in all cases) compared to other water types. Aeromonas counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. E. coli counts were higher in the vegetable crop-growing (May-October) than nongrowing (November-April) season in all water types (P < 0.05). Only one RW and both PW sites met the U.S. Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within 2 days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy setting. IMPORTANCE Increasing demands for fresh fruit and vegetables, a variable climate affecting agricultural water availability, and microbial food safety goals are pressing the need to identify new, safe, alternative sources of irrigation water. Our study generated microbial data collected over a 2-year period from potential sources of irrigation (rivers, ponds, and reclaimed water sites). Pond water was found to comply with Food Safety Modernization Act (FSMA) microbial standards for irrigation of fruit and vegetables. Bacterial counts in reclaimed water, a resource that is not universally allowed on fresh produce in the United States, generally met microbial standards or needed minimal mitigation. We detected the most seasonality and the highest microbial loads in river water, which emerged as the water type that would require the most mitigation to be compliant with established FSMA standards. This data set represents one of the most comprehensive, longitudinal analyses of alternative irrigation water sources in the United States.

scholarly journals Draft Genome Sequences of Five Neonatal Meningitis-CausingEscherichia coliIsolates (SP-4, SP-5, SP-13, SP-46, and SP-65)

2018 ◽  
Vol 6 (16) ◽  
pp. e00091-18 ◽  
Author(s):  
Aixia Xu ◽  
James R. Johnson ◽  
Shiowshuh Sheen ◽  
Christopher Sommers
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
The Netherlands ◽  
Draft Genome ◽  
Neonatal Meningitis ◽  
Genome Sequences ◽  
Processing Technologies ◽  
Content Type ◽  
E Coli

ABSTRACTNeonatal meningitis-causingEscherichia coliisolates (SP-4, SP-5, SP-13, SP-46, and SP-65) were recovered between 1989 and 1997 from infants in the Netherlands. Here, we report the draft genome sequences of these fiveE. coliisolates, which are currently being used to validate food safety processing technologies.

scholarly journals Dual-Serotype Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and O26:H11 Strains

2012 ◽  
Vol 78 (17) ◽  
pp. 6341-6344 ◽  
Author(s):  
Rong Wang ◽  
Norasak Kalchayanand ◽  
James L. Bono ◽  
John W. Schmidt ◽  
Joseph M. Bosilevac
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Potential Risk ◽  
Shiga Toxin ◽  
Biofilm Formation ◽  
Escherichia Coli O157 ◽  
Content Type ◽  
E Coli

ABSTRACTEscherichia coliO26:H11 strains were able to outgrow O157:H7 companion strains in planktonic and biofilm phases and also to effectively compete with precolonized O157:H7 cells to establish themselves in mixed biofilms.E. coliO157:H7 strains were unable to displace preformed O26:H11 biofilms. Therefore,E. coliO26:H11 remains a potential risk in food safety.

scholarly journals Stress-Induced Evolution of Heat Resistance and Resuscitation Speed in Escherichia coli O157:H7 ATCC 43888

2016 ◽  
Vol 82 (22) ◽  
pp. 6656-6663 ◽  
Author(s):  
Elisa Gayán ◽  
Alexander Cambré ◽  
Chris W. Michiels ◽  
Abram Aertsen
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Hydrostatic Pressure ◽  
Foodborne Pathogens ◽  
High Hydrostatic Pressure ◽  
Cross Resistance ◽  
Content Type ◽  
E Coli ◽  
Mild Heat ◽  
Mutant Strains

ABSTRACTThe development of resistance in foodborne pathogens to food preservation techniques is an issue of increasing concern, especially in minimally processed foods where safety relies on hurdle technology. In this context, mild heat can be used in combination with so-called nonthermal processes, such as high hydrostatic pressure (HHP), at lower individual intensities to better retain the quality of the food. However, mild stresses may increase the risk of (cross-)resistance development in the surviving population, which in turn might compromise food safety. In this investigation, we examined the evolution ofEscherichia coliO157:H7 strain ATCC 43888 after recurrent exposure to progressively intensifying mild heat shocks (from 54.0°C to 60.0°C in 0.5°C increments) with intermittent resuscitation and growth of survivors. As such, mutant strains were obtained after 10 cycles of selection with ca. 106-fold higher heat resistance than that for the parental strain at 58.0°C, although this resistance did not extend to temperatures exceeding 60.0°C. Moreover, these mutant strains typically displayed cross-resistance against HHP shock and displayed signs of enhanced RpoS and RpoH activity. Interestingly, additional cycles of selection maintaining the intensity of the heat shock constant (58.5°C) selected for mutant strains in which resuscitation speed, rather than resistance, appeared to be increased. Therefore, it seems that resistance and resuscitation speed are rapidly evolvable traits inE. coliATCC 43888 that can compromise food safety.IMPORTANCEIn this investigation, we demonstrated thatEscherichia coliO157:H7 ATCC 43888 rapidly acquires resistance to mild heat exposure, with this resistance yielding cross-protection to high hydrostatic pressure treatment. In addition, mutants ofE. coliATCC 43888 in which resuscitation speed, rather than resistance, appeared to be improved were selected. As such, both resistance and resuscitation speed seem to be rapidly evolvable traits that can compromise the control of foodborne pathogens in minimal processing strategies, which rely on the efficacy of combined mild preservation stresses for food safety.

scholarly journals Importance of Pyruvate Sensing and Transport for the Resuscitation of Viable but NonculturableEscherichia coliK-12

2018 ◽  
Vol 201 (3) ◽  
Author(s):  
Cláudia Vilhena ◽  
Eugen Kaganovitch ◽  
Alexander Grünberger ◽  
Magdalena Motz ◽  
Ignasi Forné ◽  
...  
Keyword(s):  
Response Regulator ◽  
Bacterial Species ◽  
Microfluidic System ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
E Coli ◽  
High Affinity ◽  
Vbnc Bacteria ◽  
K 12

ABSTRACTEscherichia coliand many other bacterial species can enter into a viable but nonculturable (VBNC) state, which is a survival strategy adopted by cells exposed to adverse environmental conditions. Pyruvate is known to be one factor that promotes resuscitation of VBNC cells. Here we studied the role of a pyruvate-sensing network, composed of the histidine kinase-response regulator systems BtsS/BtsR and YpdA/YpdB and the target genebtsT, encoding the high-affinity pyruvate/H+symporter BtsT, in the resuscitation of VBNCE. coliK-12 cells after exposure to cold for 120 days. Analysis of the proteome of VBNC cells revealed upregulation, relative to exponentially growing cells, of BtsT and other proteins involved in pyruvate metabolism. Provision of pyruvate stimulated protein and DNA biosynthesis, and thus resuscitation, in wild-type but notbtsSR ypdABmutant VBNC cells. This result was corroborated by time-dependent tracking of the resuscitation of individual VBNCE. colicells observed in a microfluidic system. Finally, transport assays revealed that14C-labeled pyruvate was rapidly taken up into VBNC cells by BtsT. These results provide the first evidence that pyruvate is taken up as a carbon source for the resuscitation of VBNCE. colicells.IMPORTANCEViable but nonculturable (VBNC) bacteria do not form colonies in standard medium but otherwise retain their metabolic activity and can express toxic proteins. Many bacterial genera, includingEscherichia,Vibrio, andListeria, have been shown to enter the VBNC state upon exposure to adverse conditions, such as low temperature, radiation, and starvation. Ultimately, these organisms pose a public health risk with potential implications for the pharmaceutical and food industries, as dormant organisms are especially difficult to selectively eliminate and VBNC bacteria can be resuscitated if placed in an environment with appropriate nutrition and temperature. Here we used a microfluidic system to monitor the resuscitation of single VBNC cells over time. We provide new molecular insights into the initiation of resuscitation by demonstrating that VBNCE. colicells rapidly take up pyruvate with an inducible high-affinity transporter, whose expression is triggered by the BtsSR-YpdAB sensing network.

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