Evaluation of Propidium Monoazide and Quantitative PCR To Quantify Viable Campylobacter jejuni Biofilm and Planktonic Cells in Log Phase and in a Viable but Nonculturable State

2015 ◽  
Vol 78 (7) ◽  
pp. 1303-1311 ◽  
Author(s):  
BRENDA MAGAJNA ◽  
HEIDI SCHRAFT
Keyword(s):  
Quantitative Pcr ◽  
Campylobacter Jejuni ◽  
Viable Cell ◽  
Propidium Monoazide ◽  
Vbnc State ◽  
Planktonic Cells ◽  
Viable But Nonculturable ◽  
Cell Counts ◽  
Routine Monitoring ◽  
Novel Method

Despite being considered fragile and fastidious, Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in the developed world. C. jejuni survives stresses by forming biofilms or entering a viable but nonculturable (VBNC) state. To investigate the number of viable cells in samples exposed to low nutrient and temperature stress, a novel method, propidium monoazide quantitative PCR (PMAqPCR), was compared with BacLight biovolume analysis and conventional plate counting for the enumeration of C. jejuni–removed biofilm cells and separately grown planktonic cells in late log phase (24 h). There were no significant differences between viable cell counts obtained using PMAqPCR and those from plate counts or BacLight biovolume analyses for each sample, confirming that this method provides results consistent with those from accepted enumeration methods (P > 0.05). To induce a VBNC state, C. jejuni planktonic cells and dislodged and washed biofilm cells were separately incubated in phosphate-buffered saline at 4°C for up to 60 days. Even when cells exposed to stress were provided with enrichment in Bolton broth before plating, treated biofilm cells lost culturability by day 10, whereas their planktonic counterparts remained culturable to day 60. The nonculturable biofilm cells remained viable in high numbers to day 60, and viable cell counts from the PMAqPCR (6.15 log cells per ml) were not significantly different from those obtained using the BacLight assay (6.98 log cells per ml) (P > 0.05), confirming that this novel method is also reliable for cells exposed to stress for extended periods. PMAqPCR shows promise for analysis where C. jejuni exists in biofilms or in the VBNC state. Adopting PMAqPCR in routine monitoring, in conjunction with improved biofilm cell collection methods, will allow for more accurate enumeration of viable and potentially virulent cells, leading to improved sanitation and reduced incidence of infection.

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.

Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration

2011 ◽  
Vol 87 (3) ◽  
pp. 263-272 ◽  
Author(s):  
Jessica K. van Frankenhuyzen ◽  
Jack T. Trevors ◽  
Hung Lee ◽  
Cecily A. Flemming ◽  
Marc B. Habash
Keyword(s):  
Quantitative Pcr ◽  
Pathogen Detection ◽  
Viable Cell ◽  
Propidium Monoazide ◽  
Cell Enumeration

scholarly journals Survival of Host-Associated Bacteroidales Cells and Their Relationship with Enterococcus spp., Campylobacter jejuni, Salmonella enterica Serovar Typhimurium, and Adenovirus in Freshwater Microcosms as Measured by Propidium Monoazide-Quantitative PCR

2011 ◽  
Vol 78 (4) ◽  
pp. 922-932 ◽  
Author(s):  
Sungwoo Bae ◽  
Stefan Wuertz
Keyword(s):  
Quantitative Pcr ◽  
Campylobacter Jejuni ◽  
Salmonella Enterica ◽  
Sunlight Exposure ◽  
Propidium Monoazide ◽  
Content Type ◽  
Significant Difference ◽  
Serovar Typhimurium ◽  
Dark Conditions ◽  
Realistic Information

ABSTRACTThe ideal host-associated genetic fecal marker would be capable of predicting the presence of specific pathogens of concern. Flowthrough freshwater microcosms containing mixed feces and inocula of the pathogensCampylobacter jejuni,Salmonella entericaserovar Typhimurium, and adenovirus were placed at ambient temperature in the presence and absence of diurnal sunlight. The totalEnterococcusDNA increased during the early periods (23 h) under sunlight exposure, even though cultivableEnterococcusand DNA in intact cells, as measured by propidium monoazide (PMA), decreased with first-order kinetics during the entire period. We found a significant difference in the decay of host-associatedBacteroidalescells between sunlight exposure and dark conditions (Pvalue < 0.05), whereas the persistence of host-associatedBacteroidalesDNA was comparable. The 2-log reduction times of adenovirus were 72 h for sunlight exposure and 99 h for dark conditions with similar decay rate constants (Pvalue = 0.13). The persistences of fecalBacteroidalescells andCampylobactercells exposed to sunlight were similar, and host-associatedBacteroidalesDNA and waterborne pathogen DNA were degraded at comparable rates (Pvalues > 0.05). Overall, the ratio of quantitative PCR (qPCR) cycle threshold (CT) values with and without PMA treatment was indicative of the time elapsed since inoculation of the microcosm with (i) fecal material from different animal sources based on host-associatedBacteroidalesand (ii) pure cultures of bacterial pathogens. The use of both PMA-qPCR and qPCR may yield more realistic information about recent sources of fecal contamination and result in improved prediction of waterborne pathogens and assessment of health risk.

scholarly journals Recovery in Embryonated Eggs of Viable but Nonculturable Campylobacter jejuni Cells and Maintenance of Ability To Adhere to HeLa Cells after Resuscitation

1999 ◽  
Vol 65 (11) ◽  
pp. 5154-5157 ◽  
Author(s):  
J. M. Cappelier ◽  
J. Minet ◽  
C. Magras ◽  
R. R. Colwell ◽  
M. Federighi
Keyword(s):  
Surface Water ◽  
Campylobacter Jejuni ◽  
Hela Cells ◽  
Pathogenic Bacteria ◽  
Vbnc State ◽  
Adhesion Properties ◽  
Viable But Nonculturable

ABSTRACT The existence of a viable but nonculturable (VBNC) state has been described for Campylobacter jejuni as it had been for a number pathogenic bacteria. Three C. jejuni human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC C. jejuni cells. Recovered cells kept their adhesion properties.

scholarly journals Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

2006 ◽  
Vol 73 (4) ◽  
pp. 1349-1354 ◽  
Author(s):  
Meng Du ◽  
Jixiang Chen ◽  
Xiaohua Zhang ◽  
Aijuan Li ◽  
Yun Li ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Large Population ◽  
Edwardsiella Tarda ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Cell Counts ◽  
Nonculturable State ◽  
Plate Counts ◽  
Temperature Upshift ◽  
Viable But Nonculturable State

ABSTRACT Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4�C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 109 CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.

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 Bactericidal Activity of Lactic Acid against Clinical, Carbapenem-Hydrolyzing, Multi-Drug-Resistant Klebsiella pneumoniae Planktonic and Biofilm-Forming Cells

Antibiotics ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 181 ◽  
Author(s):  
Bardhan ◽  
Chakraborty ◽  
Bhattacharjee
Keyword(s):  
Lactic Acid ◽  
Klebsiella Pneumoniae ◽  
Cell Damage ◽  
Viable Cell ◽  
World Health ◽  
Preterm Neonates ◽  
Planktonic Cells ◽  
Cell Counts ◽  
Efficacy Parameters ◽  
Health Organization

: Carbapenem resistant Klebsiella pneumoniae has been highlighted to be a critical pathogen by the World Health Organization. The objectives of this study were to assess the efficacy of lactic acid (LA) against planktonic cells and biofilms formed by carbapenem-hydrolyzing K. pneumoniae isolates obtained from the nares of preterm neonates. Time-kill assays with graded percentages of (v/v) LA in water were initially carried out against planktonic cells of a meropenem (MRP)-resistant K. pneumoniae isolate, JNM11.C4. The efficacy parameters such as optimal incubation time and minimum inhibitory concentration were determined by comparing colony-forming unit counts (log(10)CFU). Scanning electron microscopy was used to visualize cell damage. Likewise, JNM11.C4 biofilms were treated with graded series of (v/v) LA. Six carbapenem-hydrolyzing isolates were next used to validate the results. A reduction of 3.6 ± 0.6 log(10) CFU/mL in JNM11.C4 planktonic cells and >3 ± 0.03log(10) CFU/mL in biofilm-forming cells were observed using 0.225% and 2% LA, respectively, after three hours. Similar decreases in viable cell-counts were observed both in the case of planktonic (˃3.6 ± 0.3log(10) CFU/mL) and biofilm-forming cells (3.8 ± 0.3log(10) CFU/mL) across all the six clinical isolates. These results indicate that LA is an effective antimicrobial against planktonic carbapenem-hydrolyzing K. pneumoniae cells and biofilms.

scholarly journals Enumeration of Viable Listeria monocytogenes Cells by Real-Time PCR with Propidium Monoazide and Ethidium Monoazide in the Presence of Dead Cells

2007 ◽  
Vol 73 (24) ◽  
pp. 8028-8031 ◽  
Author(s):  
Y. Pan ◽  
F. Breidt
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Antimicrobial Effect ◽  
Viable Cell ◽  
Propidium Monoazide ◽  
Ethidium Monoazide ◽  
Cell Counts

ABSTRACT Propidium monoazide (PMA) and ethidium monoazide were used for enumeration of viable Listeria monocytogenes cells in the presence of dead cells. PMA had no antimicrobial effect on L. monocytogenes. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells from planktonic and biofilm sources over a 4-log range.

scholarly journals Enumeration of Viable Non-Culturable Vibrio cholerae Using Droplet Digital PCR Combined With Propidium Monoazide Treatment

2021 ◽  
Vol 11 ◽  
Author(s):  
Shuo Zhao ◽  
Jingyun Zhang ◽  
Zhe Li ◽  
Yu Han ◽  
Biao Kan
Keyword(s):  
Vibrio Cholerae ◽  
Quantitative Pcr ◽  
Bacterial Species ◽  
Accurate Method ◽  
Single Copy ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Vbnc State

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.

scholarly journals Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae

2021 ◽  
Vol 9 (5) ◽  
pp. 927
Author(s):  
Takashi Hamabata ◽  
Mitsutoshi Senoh ◽  
Masaaki Iwaki ◽  
Ayae Nishiyama ◽  
Akihiko Yamamoto ◽  
...  
Keyword(s):  
Low Temperatures ◽  
Diphtheria Toxin ◽  
Pathogenic Bacteria ◽  
Corynebacterium Diphtheriae ◽  
Human Pathogen ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Environmental Survival ◽  
Reverse Transcription Quantitative Pcr ◽  
Morphological Differences

Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.

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