Correspondence/Rebuttal pubs.acs.org/est
Comment on “UV Disinfection Induces a VBNC State in Escherichia coli and Pseudomonas aeruginosa”
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regrowth was significant and not related to deviations from nonhomogeneous bacterial aggregate solutions.
iable but nonculturable (VBNC) is described as a condition where cells lose culturability upon exposure to environmental stress, but retain their viability and potential to revert to a metabolically active and infectious state.1 These observations have generated interest in understanding the role of VBNC in water disinfection and potential public health implications. Zhang et al. presents a study on the potential of UV disinfection to induce VBNC in bacteria.2 In reviewing the study, fundamental concerns were identified with the methods used, which deviate from standard practice, calling into question their results and subsequent conclusions.
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MOLECULAR ASSAYS The authors also report a series of molecular assays performed on the bacterial culture before and after UV irradiation.2 However, there is an uncharacteristic lack of standards, quality control, and attention to detail in their methods and results.11 Figure 2 in Zhang et al.2 compares the detection of the 16s gene by long PCR (1465 bp) with short PCR (194 pb) of the same bacterial gene, reporting a 5 log difference in the untreated sample. The number of gene copies should be identical in both the untreated bacteria PCR assays and when comparing long-range qPCR with standard qPCR; any differences would be related to genome damage from UV exposure.12 In the same figure, they report cycle thresholds rather than gene copies, another indication that proper control and standardization of the qPCR assay was not performed.13 Additionally, the primers used to generate the data in Figures 3a and 4 are not identified, and in Figure 3a the legend indicates log copies but the y axis indicates inactivation rate. The observation in Figure 3 of constant expression of bacterial genes after UV irradiation should be attributed to their experimental design rather than presence of VBNC cells since the initial concentration of RNA was adjusted to 1 μg of RNA before reverse transcription. Considering the large number of surviving and metabolically active bacterial cells, the authors eliminate possible detection of any variation of measured gene expression in the nonculturable population. Furthermore, expression of gadA and oprL would not be expected to change following UV exposure because the cells were not exposed to either pH or thermal stress. Moreover, due to their persistence at time scales greater than the author’s time scale2 of 3 min, nucleic acids alone are not a good indicator of cell viability after UV treatments.14−18 Finally, the PMA-qPCR and cell staining imaging assays rely on cell membrane permeability for dye penetration and downstream quantification or imaging. Although membrane receptors and signaling pathways can be affected,19 LP UV is not known to induce membrane permeability. The conclusions from these data are overstated, especially in comparison to an oxidative treatment like chlorine. UV disinfection has been practiced for decades to protect public health and has not been implicated in any major waterborne disease event. In our opinion, given the major concerns in the scientific methods used by Zhang et al.,2 the authors are unjustifiably alarmist and inaccurate in interpreting their results to conclude that UV disinfection has “potential health risks” in spite of the extensive literature to the contrary on this subject. Karl G. Linden*,† Natalie M. Hull† Roberto A. Rodriguez‡
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UV IRRADIATION METHODOLOGY Standard methods for the practice of UV disinfection experiments are well-defined (see Bolton and Linden;3 >300 Web of Science citations) but were not followed. For instance, UV irradiance and sample absorbance were not measured on their aqueous suspended microbe samples. While they mention the characteristics of the irradiation setup, there is no mention of a method or measurement instrument, and their doses reported are an exact calculation of the reported irradiance multiplied by the time of exposure, which is not standard practice in UV bench scale collimated beam studies. The disinfection results on a UV dose basis are highly irregular. Only 2.29 log inactivation of E. coli at a dose of 50 mJ/cm2 was reported. According to compiled UV disinfection data by Hijnen et al.,4 the lower 95% confidence interval inactivation rate constant for E. coli is 0.46 cm2/mJ, indicating that a dose of 50 mJ/cm2 should result in over 23 log inactivation. Put another way, a log inactivation of 2.29 should require a dose of only 5 mJ/cm2.
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MICROBIOLOGICAL APPROACH The fact that the experiments start with an E. coli concentration of 109 CFU/mL is concerning. This high concentration of microbes can result in significant aggregation,5−7 which is apparently confirmed by the observations of the authors reporting tailing after a dose of 100 mJ/cm2. This tailing is typically attributed to the survivors being aggregated or particle associated.8,9 Zhang et al. claim resuscitation of VBNC cells after UV treatment based on regrowth observations of the bacteria in LB broth media.2 A similar experimental setup was used by Whitesides and Oliver10 where low temperature reduced 106 CFU/mL of V. vulnificus to 0 CFU/mL, and regrowth from a VBNC state was observed even after 1000 fold dilution of the sample. Interestingly, Zhang et al. never achieved 0 CFU/mL.2 The concentration of E. coli after irradiation was still ∼105 CFU/ mL, and regrowth was only observed in the initial dilutions. For both E. coli and P. aeruginosa, regrowth correlates to bacterial numbers surviving UV irradiation. Therefore, their results may be more attributable to distribution and dispersion of surviving bacteria during dilution series preparation than to resuscitation of these bacteria from a VBNC state. Additionally, no statistical analysis was performed to demonstrate that the amount of © XXXX American Chemical Society
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DOI: 10.1021/acs.est.5b02534 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Correspondence/Rebuttal
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(18) Coutard, F.; Pommepuy, M.; Loaec, S.; Hervio-Heath, D. mRNA detection by reverse transcription−PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state. J. Appl. Microbiol. 2005, 98 (4), 951−961. (19) Schwarz, T. UV light affects cell membrane and cytoplasmic targets. J. Photochem. Photobiol., B 1998, 44, 91−96.
University of Colorado Boulder, Boulder, Colorado 80309, United States ‡ The University of Texas Health Science Center at Houston, El Paso, Texas 79902, United States
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Oliver, J. D. Public Health Significance of Viable but Nonculturable Bacteria. In Non- Culturable Microorganisms in the Environment; ASM Press: Washington, DC, 2000; pp 277−300. (2) Zhang, S.; Ye, C.; Lin, H.; Lv, L.; Yu, X. UV Disinfection Induces a Vbnc State in Escherichia coli and Pseudomonas aeruginosa. Environ. Sci. Technol. 2015, 49 (3), 1721−1728. (3) Bolton, J. R.; Linden, K. G. Standardization of methods for fluence (UV dose) determination in bench-scale UV experiments. J. Environ. Eng. 2003, 129 (3), 209−215. (4) Hijnen, W. A. M.; Beerendonk, E. F.; Medema, G. J. Inactivation credit of UV radiation for viruses, bacteria, and protozoan (oo)cysts in water: a review. Water Res. 2006, 40, 3−22. (5) Stewart, M. H.; Olson, B. H. Bacterial Resistance to Potable Water Disinfectants. In Modeling Disease Transmission and Its Prevention by Disinfection; Hurst, C.J., Ed.; Cambridge University Press: Cambridge, U.K., 1996; pp 140−192. (6) Bohrerova, Z.; Linden, K. G. UV and chlorine disinfection of Mycobacterium in wastewater: impact of aggregation. Water Environ. Res. 2006, 78 (6), 565−571. (7) Mamane, H. Impact of particles on UV disinfection of water and wastewater effluents: a review. Rev. Chem. Eng. 2008, 24 (2−3), 67−157. (8) Emerick, R. W.; Loge, F. J.; Thompson, D. E.; Darby, J. L. Factors Influencing Ultraviolet Disinfection Performance Part 2: Association of Coliform Bacteria with Wastewater Particles. Water Environ. Res. 1999, 71, 1178−1187. (9) Templeton, M. R.; Andrews, R. C.; Hofmann, R. Inactivation of particle-associated viral surrogates by ultraviolet light. Water Res. 2005, 39, 3487−3500. (10) Whitesides, M. D.; Oliver, J. D. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. Appl. Environ. Microbiol. 1997, 63 (3), 1002−1005. (11) Udvardi, M. K.; Czechowski, T.; Scheible, W. R. Eleven Golden Rules of Quantitative RT-PCR. Plant Cell 2008, 20, 1736−1737. (12) Rodríguez, R. A.; Bounty, S.; Linden, K. Long-Range Quantitative PCR for Determining Inactivation of Adenovirus 2 by Ultraviolet Light. J. Appl. Microbiol. 2013, 114 (6), 1854−1865. (13) Ramakers, C.; Ruijter, J. M.; Deprez, R. H.; Moorman, A. Assumption-Free analysis of quantitative real-time polymerase chain reaction(PCR) data. Neurosci. Lett. 2003, 339, 62−66. (14) McKillip, J. L.; Jaykus, L. A.; Drake, M. rRNA Stability in HeatKilled and UV-Irradiated Enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7. Appl. Environ. Microbiol. 1998, 64 (11), 4264− 4268. (15) Sheridan, G. E. C.; Masters, C. I.; Shallcross, J. A.; Mackey, B. M. Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in Escherichia coli Cells. Appl. Environ. Microbiol. 1998, 64 (4), 1313−1318. (16) Birch, L.; Dawson, C. E.; Cornett, J. H.; Keer, J. T. A comparison of nucleic acid amplification technique for the assessment of bacterial viability. Lett. Appl. Microbiol. 2001, 33, 296−301. (17) del Mar Lleò, M.; Pierobon, S.; Tafi, M. C.; Signoretto, C.; Canepari, P. mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm. Appl. Environ. Microbiol. 2000, 66 (10), 4564−4567. B
DOI: 10.1021/acs.est.5b02534 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
