A Microbiological Paradox: Viable but Nonculturable Bacteria with Special Reference to Vibrio cholerae

1996 ◽  
Vol 59 (1) ◽  
pp. 96-101 ◽  
Author(s):  
ANWARUL HUQ ◽  
RITA R. COLWELL
Keyword(s):  
Vibrio Cholerae ◽  
Culture Media ◽  
Direct Detection ◽  
Bacterial Species ◽  
Detection Methods ◽  
Bacterial Cells ◽  
Culture Methods ◽  
Viable But Nonculturable ◽  
Bacteriological Culture ◽  
Nonculturable State

The observation that directly-detectable bacterial cells are unable to grow on bacteriological culture media under certain conditions raises questions regarding the viability of these cells. Various terminologies have been used to describe substrate-responsive and metabolically-active bacterial cells that cannot be cultured. The currently-accepted term is “viable but nonculturable.” During the past 15 years, the viable but nonculturable phenomenon has been actively investigated. Bacterial pathogens in the viable but nonculturable state can maintain virulence and produce disease. These organisms may escape detection if bacteriological culture methods are solely used. Thus, methods for direct detection of specific pathogens in food, water and environmental samples are preferable. Viable but nonculturable Vibrio cholerae have been extensively studied, and several sensitive and reliable direct-detection kits have been developed. Viable but nonculturable forms of bacteria are now recognized as a common phenomenon, observable in many bacterial species, which suggests that standard bacteriological laboratory protocols for assessing microbiological safety of food and drinking water are less reliable than direct detection methods.

scholarly journals Temperature-induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation?

Microbiology ◽  
1995 ◽  
Vol 141 (2) ◽  
pp. 377-383 ◽  
Author(s):  
J. Ravel ◽  
I. T. Knight ◽  
C. E. Monahan ◽  
R. T. Hill ◽  
R. R. Colwell
Keyword(s):  
Vibrio Cholerae ◽  
Viable But Nonculturable ◽  
Nonculturable State ◽  
State Growth ◽  
Viable But Nonculturable State

scholarly journals Specific and Rapid Enumeration of Viable but Nonculturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry

2010 ◽  
Vol 76 (15) ◽  
pp. 5088-5096 ◽  
Author(s):  
Mohiuddin M. Taimur Khan ◽  
Barry H. Pyle ◽  
Anne K. Camper
Keyword(s):  
Flow Cytometry ◽  
Pseudomonas Syringae ◽  
Phase 1 ◽  
Epifluorescence Microscopy ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Culture Methods ◽  
Viable But Nonculturable ◽  
Gram Negative ◽  
Analytical Technique

ABSTRACT An issue of critical concern in microbiology is the ability to detect viable but nonculturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods) are also biased and time-consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella enterica serovar Typhimurium cells after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40, and propidium iodide (PI). The FCM data were compared with those for specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were nonculturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell membranes and were therefore theoretically dead. Data obtained using four different Gram-negative bacteria exposed to heat and stained with PI also illustrate the usefulness of the approach for the rapid and unbiased detection of dead versus live organisms.

scholarly journals A distributed computational search strategy for the identification of diagnostics targets: Application to finding aptamer targets for methicillin-resistant staphylococci

2014 ◽  
Vol 11 (2) ◽  
pp. 80-92 ◽  
Author(s):  
Keith Flanagan ◽  
Simon Cockell ◽  
Colin Harwood ◽  
Jennifer Hallinan ◽  
Sirintra Nakjang ◽  
...  
Keyword(s):  
Direct Detection ◽  
Bacterial Species ◽  
Distributed Databases ◽  
Bacterial Genome ◽  
Cost Effective ◽  
Pcr Primers ◽  
Detection Methods ◽  
Data Sets ◽  
Peptide Aptamers ◽  
Automated Processing

Summary The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

Sampling efficiency of Candida auris from healthcare surfaces: culture and nonculture detection methods

2021 ◽  
pp. 1-3
Author(s):  
William A. Furin ◽  
Lisa H. Tran ◽  
Monica Y. Chan ◽  
Amanda K. Lyons ◽  
Judith Noble-Wang ◽  
...  
Keyword(s):  
Esterase Activity ◽  
Detection Methods ◽  
Sampling Efficiency ◽  
Activity Assay ◽  
Candida Auris ◽  
Viable But Nonculturable ◽  
Nonculturable State ◽  
Plastic Surfaces ◽  
Viable But Nonculturable State

Abstract Sponges and swabs were evaluated for their ability to recover Candida auris dried 1 hour on steel and plastic surfaces. Culture recovery ranged from <0.1% (sponges) to 8.4% (swabs), and cells detected with an esterase activity assay revealed >50% recovery (swabs), indicating that cells may enter a viable but nonculturable state.

scholarly journals Frequency of Vibrio cholerae in the Water and Plankton Samples of South- Western Coastal Aquatic Habitats of Bangladesh

2012 ◽  
Vol 36 (1) ◽  
pp. 71-78 ◽  
Author(s):  
Md Mansurul Haque ◽  
Munirul Alam ◽  
Abdus Salam
Keyword(s):  
Vibrio Cholerae ◽  
Culture Media ◽  
The Other ◽  
Environmental Surveillance ◽  
Aquatic Habitats ◽  
Culture Methods ◽  
Counting Technique ◽  
Other Hand ◽  
Investigation Period ◽  
Academy Of Sciences

Monthly environmental surveillance was carried out for three consecutive years in the coastal aquatic habitats of Mathbaria, with a view to assessing the culturability and abundance of V. cholerae. The study revealed that 93 samples (29%) out of 324 were positive for V. cholerae O1 on TCBS and TTGA culture media but no sample was found positive for V. cholerae O139 in the same culture media. On the other hand, all the water and plankton samples were found positive for V. cholerae O1 and O139 in DFA counting technique. Similarly, V. cholerae non-O1 and non- O139 were detected from all samples through culture methods throughout the investigation period.   V. cholerae O139 has been found to be absolutely non-responsive to artificial enrichment and culture media with the advancement of time. Therefore, water and plankton samples can also be concluded to be the additional reservoir of V. cholerae DOI: http://dx.doi.org/10.3329/jbas.v36i1.10922 Journal of Bangladesh Academy of Sciences, Vol. 36, No. 1, 71-78, 2012

Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters

1992 ◽  
Vol 38 (5) ◽  
pp. 365-369 ◽  
Author(s):  
Gary A. Toranzos ◽  
Abdiel J. Alvarez
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Culture Media ◽  
Direct Detection ◽  
Bacterial Cells ◽  
Chain Reaction ◽  
Detection And Identification ◽  
Low Concentrations ◽  
Polymerase Chain

The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible. These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification. We have developed an assay for the detection of bacterial cells in large volumes of water. Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction. This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell. This technique could be used for the detection of any bacteria or virus in water or air. Key words: polymerase chain reaction, waterborne pathogens, water.

scholarly journals Critical Assessment of Methods to Quantify Biofilm Growth and Evaluate Antibiofilm Activity of Host Defence Peptides

Biomolecules ◽  
2018 ◽  
Vol 8 (2) ◽  
pp. 29 ◽  
Author(s):  
Evan Haney ◽  
Michael Trimble ◽  
John Cheng ◽  
Quentin Vallé ◽  
Robert Hancock
Keyword(s):  
Bacterial Species ◽  
Growth Conditions ◽  
Host Defence ◽  
Detection Methods ◽  
Antibiofilm Activity ◽  
Bacterial Cells ◽  
Media Composition ◽  
Microscopy Imaging ◽  
Biofilm Growth ◽  
Tetrazolium Chloride

Biofilms are multicellular communities of bacteria that can adhere to virtually any surface. Bacterial biofilms are clinically relevant, as they are responsible for up to two-thirds of hospital acquired infections and contribute to chronic infections. Troublingly, the bacteria within a biofilm are adaptively resistant to antibiotic treatment and it can take up to 1000 times more antibiotic to kill cells within a biofilm when compared to planktonic bacterial cells. Identifying and optimizing compounds that specifically target bacteria growing in biofilms is required to address this growing concern and the reported antibiofilm activity of natural and synthetic host defence peptides has garnered significant interest. However, a standardized assay to assess the activity of antibiofilm agents has not been established. In the present work, we describe two simple assays that can assess the inhibitory and eradication capacities of peptides towards biofilms that are formed by both Gram-positive and negative bacteria. These assays are suitable for high-throughput workflows in 96-well microplates and they use crystal violet staining to quantify adhered biofilm biomass as well as tetrazolium chloride dye to evaluate the metabolic activity of the biofilms. The effect of media composition on the readouts of these biofilm detection methods was assessed against two strains of Pseudomonas aeruginosa (PAO1 and PA14), as well as a methicillin resistant strain of Staphylococcus aureus. Our results demonstrate that media composition dramatically alters the staining patterns that were obtained with these dye-based methods, highlighting the importance of establishing appropriate biofilm growth conditions for each bacterial species to be evaluated. Confocal microscopy imaging of P. aeruginosa biofilms grown in flow cells revealed that this is likely due to altered biofilm architecture under specific growth conditions. The antibiofilm activity of several antibiotics and synthetic peptides were then evaluated under both inhibition and eradication conditions to illustrate the type of data that can be obtained using this experimental setup.

scholarly journals Does Enterohemorrhagic Escherichia coliO157:H7 Enter the Viable but Nonculturable State in Salted Salmon Roe?

2000 ◽  
Vol 66 (12) ◽  
pp. 5536-5539 ◽  
Author(s):  
Sou-Ichi Makino ◽  
Tsutomu Kii ◽  
Hiroshi Asakura ◽  
Toshikazu Shirahata ◽  
Tetsuya Ikeda ◽  
...  
Keyword(s):  
Viable Cell ◽  
Bacterial Cells ◽  
Fluorescent Staining ◽  
Viable But Nonculturable ◽  
Viable Cells ◽  
Recovery System ◽  
Nonculturable State ◽  
Almost All ◽  
Viable But Nonculturable State

ABSTRACT An outbreak caused by salted salmon roe contaminated with enterohemorrhagic Escherichia coli O157 occurred in Japan in 1998. Since about 0.75 to 1.5 viable cells were estimated to cause infection, we presumed that O157 might enter the viable but nonculturable (VNC) state in salted salmon roe and consequently that viable cell numbers might be underestimated. Although patient-originating O157 cells could not grow on agar plates after 72 h of incubation in 13% NaCl, they were resuscitated in yeast extract broth, and more than 90% of the cells were shown to be viable by fluorescent staining, suggesting that almost all of them could enter the VNC state in NaCl water. Roe-originating O157 was resistant to NaCl because it could grow on agar after 72 h of incubation in NaCl water, but about 20% of cells appeared to enter the VNC state. Therefore, germfree mice were infected with O157 to examine the resuscitation of cells in the VNC state and the retention of pathogenicity. O157 that originated in roe, but not patients, killed mice and was isolated from the intestine. However, these isolates had become sensitive to NaCl. O157 cells of roe origin incubated in normal media also killed mice and were isolated from the intestine, but they also became transiently NaCl sensitive. We therefore propose that bacterial cells might enter the VNC state under conditions of stress, such as those encountered in vivo or in high salt concentrations, and then revive when those conditions have eased. If so, the VNC state in food is potentially dangerous from a public health viewpoint and may have to be considered at the time of food inspection. Finally, the establishment of a simple recovery system for VNC cells should be established.

scholarly journals Toxigenic Vibrio cholerae in the Aquatic Environment of Mathbaria, Bangladesh

2006 ◽  
Vol 72 (4) ◽  
pp. 2849-2855 ◽  
Author(s):  
Munirul Alam ◽  
Marzia Sultana ◽  
G. Balakrish Nair ◽  
R. Bradley Sack ◽  
David A. Sack ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Aquatic Environment ◽  
Fluorescent Antibody ◽  
Culture Methods ◽  
Viable But Nonculturable ◽  
Large Numbers ◽  
Direct Fluorescent Antibody ◽  
Nonculturable Cells ◽  
Seasonal Epidemics

ABSTRACT Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.

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.

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