The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria.

Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.

Desiccation-induced viable but nonculturable state in Pseudomonas putida KT2440, a survival strategy.

The potential of Pseudomonas putida KT2440 to act as a plant-growth promoter or as a bioremediator of toxic compounds can be affected by desiccation. In the present work, the bacterial survival ratio (BSR) in response to air desiccation was evaluated for P. putida KT2440 in the presence of different protectors. The BSR in the presence of nonreducing disaccharides, such as trehalose, was high after 15 days of desiccation stress (occurring at 30°C and 50% relative humidity), whereas in the absence of a protector the bacterial counts diminished to nondetectable numbers (ca 2.8 log CFU/mL). The LIVE/DEAD staining method showed that bacteria protected with trehalose maintained increased numbers of green cells after desiccation while cells without protection were all observed to be red. This indicated that nonprotected bacteria had compromised membrane integrity. However, when nonprotected bacteria subjected to 18 days of desiccation stress were rehydrated for a short time with maize root exudates or for 48 h with water (prolonged rehydration), the bacterial counts were as high as that observed for those not subjected to desiccation stress, suggesting that the cells entered the viable but nonculturable (VBNC) state under desiccation and that they returned to a culturable state after those means of rehydration. Interestingly an increase in the green color intensity of cells that returned to a culturable state was observed using LIVE/DEAD staining method, indicating an improvement in their membrane integrity. Cellular activity in the VBNC state was determined. A GFP-tagged P. putida strain expressing GFP constitutively was subjected to desiccation. After 12 days of desiccation, the GFP-tagged strain lost culturability, but it exhibited active GFP expression, which in turn made the cells green. Furthermore, the expression of 16S rRNA, rpoN (housekeeping), mutL, mutS (encoding proteins from the mismatch repair complex), and oprH (encoding an outer membrane protein) were examined by RT-PCR. All evaluated genes were expressed by both types of cells, culturable and nonculturable, indicating active molecular processes during the VBNC state.

Cuantificación de bacterias cultivables mediante el método de Goteo en Placa por Sellado (o estampado) Masivo / Quantification of cultivable bacteria by the Massive Stamping Drop Plate method

Cuando se desea cuantificar el número de bacterias presentes en múltiples muestras, los procedimientos de rutina suelen consumir mucho tiempo. En ese periodo las muestras podrían sufrir modificaciones en su población. En el presente trabajo se evaluó una metodología alternativa para cuantificar bacterias cultivables de forma masiva, rápida y económica en la que se implica el sellado, estampado o impresión de diluciones seriadas de muestras de diversa procedencia. El tiempo requerido para preparar 22 muestras para su sellado en placa es de 15 minutos. El método se basó en realizar diluciones seriadas (base 10) de las muestras líquidas originales contenidas en una placa multipozos con la ayuda de una pipeta multicanal. Después, con un replicador se tomó un volumen (aproximadamente 1,65 µl) de muestra de cada pozo, que se inoculó por sellado en un medio de crecimiento gelificado de interés. Las placas se incubaron el tiempo necesario, se contó el número de colonias presentes en la dilución contable y se calculó el número de Unidades Formadoras de Colonia por mililitro (UFC/ml) para cada muestra. La metodología se denominó "Goteo por Sellado en Placa Masivo" (GSPM) y ha sido aplicada para cuantificar exitosamente bacterias provenientes de diferentes muestras de laboratorio, por ejemplo, de cultivos líquidos, muestras clínicas (como exudados y secreciones) y bacterias presentes en la rizósfera de plantas de maíz. Sin embargo, la metodología GSPM podría aplicarse para contabilizar masivamente a bacterias de cualquier otra procedencia.

In an attempt to quantify the number of bacteria present in a high number of samples, routine procedures are usually very time-consuming. During this period of time, bacterial population could be modified. In this work, an alternative for a massive, quick and economic method was evaluated in order to count viable bacteria, consisting in the sealing or stamping of serial dilutions performed in samples from different origins. The time required to prepare 22 samples for plate stamping is only 15 minutes. The quantification was based in performing serial dilutions (10-fold) of the original liquid samples contained in a multiwell plate using a multichannel micropipette. Afterwards, using a replicator, the same volume of each sample (approximately 1,65 µl) was recovered from each well, and then it was inoculated and sealed in a solid growth media of interest. Plates were incubated as needed, colonies were counted in the quantifiable dilution and Colony Forming Units per milliliter (CFU/ml) was calculated for each sample. We called this method "Massive Stamping Drop Plate" (MSDP) and it has been successfully applied to count bacteria from different lab samples, including liquid cultures, clinical samples (exudates and secretions) and bacteria recovered from the rhizosphere of corn plants. However, MSDP could also be applied to massively count bacteria from any other source.