Correspondence/Rebuttal pubs.acs.org/est
Response to Comment on “UV Disinfection Induces a VBNC State in Escherichia coli and Pseudomonas aeruginosa” Different fluorescent dyes were adopted in amplifying short and long PCR products, i.e., sybrgreen for short and evagreen for long fragmens, respectively. This is because PCR may sometimes present false negative results when used for detecting DNA damage. So it is necessary to test more primer combinations to make the conclusion confirmed. In Figure 3b, we used the Ct value to reflect the relative gene copy numbers. The purpose was to know the changing trend of long/short gadA gene fragments of E. coli under different UV dose based on qPCR. In fact, we think the trends are more important than the specific numerical values in microbiology and water treatment research. The primers used for Figure 3a and 4 were identical. It was a clerical error that in Figure 3a the legend indicates log copies but the y axis indicates inactivation rate. Dr. Linden and his colleagues thought that the observation in Figure 3 of constant expression of bacterial genes after UV irradiation should be attributed to our experimental design rather than presence of VBNC cells. In fact, not only molecular methods but also culturing methods were tested in this study. The results from these two tests could provide rather good support to each other. The HPC results demonstrated that the culturable cell numbers could reduce with the UV dose increasing, and the RT-qPCR results remained constant, which could already demonstrate the viable but nonculturable property. In other words, UV could induce bacteria into the VBNC state. Certainly, the pure VBNC cell would make our conclusion more persuasive. Our future research will focus on the pure VBNC bacteria. Dr. Linden put forward that “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”. But this supports precisely our opinion, i.e., the UV irradiation has health risk potential because it cannot reduce the virulent ability of the pathogens. Finally, the doubt about PMA-qPCR and cell staining and imaging assays showed that Dr. Linden did not grasp our idea. What we wanted to demonstrate was that since these UV doses could not destroy the cell membrane, PMA-qPCR was not suitable for the detection of the VBNC cell after UV treatment, while another frequently used disinfectant, namely, chlorine could destroy the cell membrane. Hence, UV radiation could induce bacteria into the VBNC state much easier than chlorine. We have demonstrated in our response to another comment8 that the UV usage history was still short, and the precedent good performance could not ensure the safety forever. As scientists focusing on water quality safety assessment, we are responsible to remind industries, governments, and consumers about any health safety risk that may be brought by any water treatment technology including but not limited to UV disinfection. We want to declare again that absolute safety is
W
e thank Dr. Linden and his colleagues very much for their comments on our work about the bacteria VBNC state induced by UV radiation, although most of them were unreasonable. We are willing to discuss the questions in their comments and clarify the concerned facts here. First, UV irradiation is not high-tech; there are many published references on this issue. Although we did not follow a certain paper with more than 300 citations, we guarantee the validity of the UV disinfection method we adopted; it is sufficient. There are many papers that have been cited more than 300 times; it is impossible to cite everyone. In fact, our UV radiation installation and dose calculation method were in agreement with many other references1−3 and also in accord with the description by Bolton and Linden.4 We even adopted “random order” to ensure data precision. Although we have not corrected factors as mentioned by Bolton and Linden, the equipment and calculation methods were able to reveal the potential health risk originated from UV treatment. In Dr. Linden’s comments, there was a critical datum mentioned, i.e., the lower 95% confidence interval inactivation rate constant for E. coli is 0.46 cm2/mJ. However, we cannot find it in the reference he cited.5 We have no interest to guess why. However, is it honest to doubt others’ research using a nonexistent figure? In fact, different strains may have a completely different resistance to disinfection.6 The E. coli strain used in our study was CMCC 44103,7 which is different from those in the references cited by Dr. Linden. This may, at least partly, explain the resistance of the bacteria shown in our study. Hence, Dr. Linden’s query about our UV irradiation methodology is groundless. Dr. Linden also had questions about our initial E. coli concentration of 109 CFU/mL. He also mentioned the “tailing” after a dose of 100 mJ/cm2. We have answered this question in the response to another comment.8 In the response, we proposed the potential risk of UV disinfection in real application scenarios if the microbial aggregates and biofilm debris attached to particles were considered. When it comes to resuscitation, it should be more convincible if we started at a culturable cell density below 0 CFU/mL. However, the protocol of our series dilution was completely in agreement with Whitesides and Oliver,9 although the original HPC concentration in resuscitation experiment was 105 CFU/mL. It is important to note that the bacterial growth was observed not only at the dilution level of 10−1 CFU/mL but also at 10−2 CFU/mL, which still suggested a high possibility of the occurrence of resuscitation, although it should be with a very low frequency. The molecular biological methods we used were all frequently used ones. The standards and many notes mentioned by Dr. Linden were all general knowledge for microbiologists and molecular biologists, which were not necessarily elucidated in detail in the text. Does Dr. Linden think that the generation mechanism of UV must be explained in his paper about UV disinfection? © 2015 American Chemical Society
Published: August 13, 2015 10752
DOI: 10.1021/acs.est.5b03757 Environ. Sci. Technol. 2015, 49, 10752−10753
Environmental Science & Technology
Correspondence/Rebuttal
essential for drinking water. The results in our study were obtained through repeatable tests, and the main conclusion is unshakable.
Shenghua Zhang Chengsong Ye Wenfang Lin Xin Yu*
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Institute of Urban Environment, Chinese Academy of Science, Xiamen, 361021, P. R. China
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Loge, F. J.; Emerick, R. W.; Thompson, D. E.; Nelson, D. C.; Darby, J. L. Factors influencing ultraviolet disinfection performance part I: light penetration to wastewater particles. Water Environ. Res. 1999, 71 (3), 377−381. (2) Shin, G. A.; Linden, K. G.; Arrowood, M. J.; Sobsey, M. D. Low presssure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 2001, 67 (7), 3029− 3032. (3) Zimmer, J. L.; Slawson, R. M. Potential repair of Escherichia coli DNA following exposure to UV radiation from both medium-and lowpressure UV sources used in drinking water treatment. Appl. Environ. Microbiol. 2002, 68 (7), 3293−3299. (4) 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. (5) 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. (6) Sommer, R.; Lhotsky, M.; Hairder, T.; Cabaj, A. UV inactivation, liquid-holding recovery, and photoreaction of E. coli OH157 and other pathogenic E. coli strains in water. J. Food Prot. 2000, 63 (8), 1015− 1020. (7) 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. (8) Yu, X.; Zhang, S.; Ye, C.; Lin, W.; Lin, H.; Lv, L. Response to Comment on “UV Disinfection Induces a Vbnc State in Escherichia coli and Pseudomonas aeruginosa. Environ. Sci. Technol. 2015, 49 (12), 7502−7503. (9) Whitesides, M. D.; Oliver, J. D. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. Appl. Environ. Microbiol. 1997, 63 (3), 1002−1005.
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DOI: 10.1021/acs.est.5b03757 Environ. Sci. Technol. 2015, 49, 10752−10753
