Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed.

BackgroundLegionella pneumophila (Lp) is a water-borne opportunistic pathogen. In water, Lp can survive for an extended period of time until it encounters a permissive host. Therefore, identifying genes that are required for survival in water may help develop strategies to prevent Legionella outbreaks.ResultsWe compared the global transcriptomic response of Lp grown in a rich medium to that of Lp exposed to an artificial freshwater medium (Fraquil) for 2, 6 and 24 hours. We uncovered successive changes in gene expression required for the successful adaptation to a nutrient-limited water environment. The repression of major pathways involved in cell division, transcription and translation, suggests that Lp enters a quiescent state in water. The induction of flagella associated genes (flg, fli and mot), enhanced-entry genes (enh) and some Icm/Dot effector genes suggests that Lp is primed to invade a suitable host in response to water exposure. Moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that Lp may be more tolerant to these stresses in water. Indeed, Lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than Lp cultured in rich medium. In addition, the bdhA gene, involved in the degradation pathway of the intracellular energy storage compound polyhydroxybutyrate, is also highly expressed in water. Further characterization show that expression of bdhA during short-term water exposure is dependent upon RpoS, which is required for the survival of Lp in water. Deletion of bdhA reduces the survival of Lp in water at 37 °C.ConclusionsThe increase of antibiotic resistance and the importance of bdhA to the survival of Lp in water seem consistent with the observed induction of these genes when Lp is exposed to water. Other genes that are highly induced upon exposure to water could also be necessary for Lp to maintain viability in the water environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1869-6) contains supplementary material, which is available to authorized users.

The bacterium Legionella pneumophila is capable of intracellular replication within freshwater protozoa as well as human macrophages, the latter of which results in the serious pneumonia Legionnaires' disease. A primary factor involved in these host cell interactions is the Dot/Icm Type IV secretion system responsible for translocating effector proteins needed to establish and maintain the bacterial replicative niche. Several regulatory factors have been identified to control the expression of the Dot/Icm system and effectors, one of which is the CpxRA two-component system, suggesting essentiality for virulence. In this study, we generated cpxR, cpxA and cpxRA in-frame null mutant strains to further delineate the role of the CpxRA system in bacterial survival and virulence. We found that cpxR is essential for intracellular replication within Acanthamoeba castellanii, but not in U937-derived macrophages. Transcriptome analysis revealed that CpxRA regulates a large number of virulence-associated proteins including Dot/Icm effectors as well as Type II secreted substrates. Furthermore, the cpxR and cpxRA mutant strains were more sodium resistant than the parental strain Lp02, and cpxRA expression reaches maximal levels during postexponential phase. Taken together, our findings suggest the CpxRA system is a key contributor to L. pneumophila virulence in protozoa via virulence factor regulation.

Despite the importance of latent effects in marine invertebrates, no study has considered how larval exposure to hypoxia may have lasting impacts on the individual in later life history stages. In this study, we exposed larvae of the gastropod Crepidula onyx to 2, 3, and 6 mg O 2 l −1 , measured the larval size and total lipid content, transplanted the newly settled individuals to the field, and finally, determined the growth rate, dry weight, and filtration rate of the juveniles after 2 wk in the field. The experiments were conducted separately at low and high larval food concentrations. Under the low food concentration, both hypoxic treatments increased the time needed for the larvae to be competent to metamorphose. When they did, however, they had a size and total lipid content similar to the control larvae. The latent effects of early hypoxia exposure on juvenile growth were evident. The juvenile growth rate was reduced by ca. 14 and 10% in the 2 and 3 mg O 2 l −1 treatments, respectively. The mean dry weight and filtration rate were also significantly reduced in the hypoxic treatments. However, there was no discernible effect on larvae or juveniles when the food concentration during the larval stage was doubled, suggesting that the latent effects of hypoxia can be offset by larval access to high algal concentration. This study suggests that periodic hypoxic events and the resulting exposure of organisms to hypoxia during their early development may have effects that persist across the life history.

The water-borne pathogen Legionella pneumophila (Lp) strongly expresses the lpg1659 gene in water. This gene encodes a hypothetical protein predicted to be a membrane protein using in silico analysis. While no conserved domains were identified in Lpg1659, similar proteins are found in many Legionella species and other aquatic bacteria. RT-qPCR showed that lpg1659 is positively regulated by the alternative sigma factor RpoS, which is essential for Lp to survive in water. These observations suggest an important role of this novel protein in the survival of Lp in water. Deletion of lpg1659 did not affect cell morphology, membrane integrity or tolerance to high temperature. Moreover, lpg1659 was dispensable for growth of Lp in rich medium, and during infection of the amoeba Acanthamoeba castellanii and of THP-1 human macrophages. However, deletion of lpg1659 resulted in an early loss of culturability in water, while over-expression of this gene promoted the culturability of Lp. Therefore, these results suggest that lpg1659 is required for Lp to maintain culturability, and possibly long-term survival, in water. Since the loss of culturability observed in the absence of Lpg1659 was complemented by the addition of trace metals into water, this membrane protein is likely a transporter for acquiring essential trace metal for maintaining culturability in water and potentially in other metal-deprived conditions. Given its role in the survival of Lp in water, Lpg1659 was named LasM for Legionella aquatic survival membrane protein.

The water-borne Gram-negative bacterium Legionella pneumophila (Lp) is the causative agent of Legionnaires’ disease. Lp is typically transmitted to humans from water systems, where it grows inside amoebae. Survival of Lp in water is central to its transmission to humans. A transcriptomic study previously identified many genes induced by Lp in water. One such gene, lpg2524, encodes a putative LuxR family transcriptional regulator. It was hypothesized that this gene could be involved in the survival of Lp in water. Deletion of lpg2524 does not affect the growth of Lp in rich medium, in the amoeba Acanthamoeba castellanii, or in human macrophage-like THP-1 cells, showing that Lpg2524 is not required for growth in vitro and in vivo. Nevertheless, deletion of lpg2524 results in a faster colony-forming unit (CFU) reduction in an artificial freshwater medium, Fraquil, indicating that Lpg2524 is important for Lp to survive in water. Overexpression of Lpg2524 also results in a survival defect, suggesting that a precise level of this transcriptional regulator is essential for its function. However, our result shows that Lpg2524 is dispensable for survival in water when Lp is at a high cell density (109 CFU/mL), suggesting that its regulon is regulated by another regulator activated at high cell density.

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