Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Effectiveness of treatment of bedding and feces of laboratory animal with ozone

  • Jiao-Jiao Qiao,

    Roles Data curation

    Affiliations Department of Clinical Laboratory, Xiang-Ya Hospital, Central South University, Changsha, China, Center for the Study of Laboratory Animals, Xiang-Ya Hospital, Central South University, Changsha, China

    ⨯
  • Shan-Ni Wang,

    Roles Data curation

    Affiliations Department of Clinical Laboratory, Xiang-Ya Hospital, Central South University, Changsha, China, Center for the Study of Laboratory Animals, Xiang-Ya Hospital, Central South University, Changsha, China

    ⨯
  • Jing-Jing Li,

    Roles Data curation

    Affiliations Department of Clinical Laboratory, Xiang-Ya Hospital, Central South University, Changsha, China, Center for the Study of Laboratory Animals, Xiang-Ya Hospital, Central South University, Changsha, China

    ⨯
  • Li-Yu Chen,

    Roles Data curation

    Affiliation Department of Microbiology, Xiang-Ya School of Medicine, Central South University, Changsha, China

    ⨯
  • Mei-Mei Wang,

    Roles Data curation

    Affiliations Department of Clinical Laboratory, Xiang-Ya Hospital, Central South University, Changsha, China, Center for the Study of Laboratory Animals, Xiang-Ya Hospital, Central South University, Changsha, China

    ⨯
  • Bin Yi,

    Roles Resources

    Affiliation Department of Clinical Laboratory, Xiang-Ya Hospital, Central South University, Changsha, China

    ⨯
  • Qing-Xia Liu,

    Roles Data curation

    Affiliation Department of Clinical Laboratory, Xiang-Ya Hospital, Central South University, Changsha, China

    ⨯
  • Yun-Bo Liu,

    Roles Funding acquisition

    Affiliation Institute of Laboratory Animal Sciences, CAMS & PUMC, Beijing, China

    ⨯
  • Chen Zhang,

    Roles Funding acquisition

    Affiliation Department of Neurobiology, Beijing Key Laboratory of Neural Regeneration and Repair, School of Basic Medical Sciences, Capital Medical University, Beijing, China

    ⨯
  • Paul Honess,

    Roles Formal analysis, Writing – review & editing

    Affiliation School of Veterinary Medicine and Science, University of Nottingham, Nottingham, United Kingdom

    ⨯
  • Chang-Qing Gao

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Writing – original draft, Writing – review & editing

    cgao851@126.com

    Affiliations Department of Clinical Laboratory, Xiang-Ya Hospital, Central South University, Changsha, China, Center for the Study of Laboratory Animals, Xiang-Ya Hospital, Central South University, Changsha, China

    ⨯

Abstract

Background

The incineration and burying of the soiled bedding of laboratory animals, as well as using detergents to treat their feces, is hazardous to the environment. This highlights the need for an alternative, environmentally friendly solution for the treatment of the waste of laboratory animal facilities. This study aims to evaluate the efficacy of ozone disinfection of the soiled bedding and feces of laboratory animals.

Methods

Two grams of soiled beddings were randomly sampled from the cages of mice and rats. These samples were mixed in a beaker with 40ml saline. Ozone was piped into the beaker at a concentration of 500mg/h. Samples were taken from the beaker at time 0min, 30min, 45min and 60min after ozone treatment for microbiological culturing in an incubator for 48h. Colony form unit of each plate (CFU/plate) at each time point were counted, the mean CFU/plate at each time point after ozone treatment were compared with that present at time zero. Feces of rabbits and dogs were treated and pathogens were counted the similar way as that of bedding of the mice and rats; samples being taken at 0min, 15min, 30min, 45min and 60min.

Results

Pathogens were observed in beddings of both mice and rats as well as in feces of rabbits and dogs. Ozone treatment for 30min killed more than 93% of pathogens in the bedding of the two rodent species and 60min of treatment killed over 99% of pathogens. Treatment of rabbit and dog feces for 30min killed over 96% pathogens present, and 60min’s treatment killed nearly all the pathogens. Both Gram positive and Gram negative pathogens were sensitive to ozone treatment.

Conclusion

Ozone treatment of bedding and feces is an effective and environment friendly way to deal with the waste of animal facilities, saving energy and potentially enabling their reuse as fertilizer.

Citation: Qiao J-J, Wang S-N, Li J-J, Chen L-Y, Wang M-M, Yi B, et al. (2022) Effectiveness of treatment of bedding and feces of laboratory animal with ozone. PLoS ONE 17(4): e0266223. https://doi.org/10.1371/journal.pone.0266223

Editor: Qiang Ding, University of Alabama at Birmingham, UNITED STATES

Received: January 26, 2022; Accepted: March 16, 2022; Published: April 6, 2022

Copyright: © 2022 Qiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the following OSF repository: https://osf.io/xgt3q/.

Funding: This study was supported by grants from the National Key Research and Development Program of China (2017YFA0105201 to Y.B.L and C. Z) and a grant from Science and Technology Department of Hunan Province (2021JJ31040 to C.Q.G).

Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

1. Introduction

Cage bedding is one of the important factors that can influence the microenvironment of laboratory animals, and thus it may also impact on experimental results. Numerous studies have been conducted to improve the quality of bedding materials, but studies on how to treat soiled bedding before disposal are scarce [1–4]. The disposal of laboratory animal waste is an important issue as large amounts of soiled bedding, which contains not only excreta but potentially also tested compounds, from research and breeding facilities has potentially significant environmental impact. To date there seem to be no guidelines on with the safe and environmentally sensitive disposal of soiled bedding and other laboratory animal waste. Currently, in China, most soiled bedding is either incinerated or deep buried. Both methods are environment unfriendly.

Miyamoto et al. [5, 6] demonstrated that soiled bedding can be recycled for repeated use by applying soft hydrothermal processing. This processing not only enables reuse of the bedding, but actually improves the bedding’s capability to reduce in-cage levels of particulates and ammonia [6]. However, the process the authors describe is complicated, involving multiple machines and occupying considerable space, which is often very limited in animal facilities and this challenges the practical application of the proposed bedding treatment system.

Excreta of laboratory animals, including that of rabbits and dogs, are currently treated with chemical agents, such as those containing chlorine, which are typically hazardous to the environment. In short, the current treatment methods for animal waste, such as bedding and feces, are not optimal and need further investigation and better solutions.

Ozone has long been used to disinfect water and air, as well as other materials [7–11]. We therefore hypothesize that ozone treatment should be effective for inactivating pathogens in bedding and feces. Therefore, the aim of this study is to assess the effectiveness of ozone treatment for disinfecting soiled bedding and feces from laboratory animals, potentially enabling the use of this waste as fertilizer instead of its disposal via incineration or burying; thereby saving energy and reducing environmental impact.

2. Materials and methods

2.1 Bedding material sampling and treatment with ozone

Five mouse cages and five rat cages were randomly selected from a laboratory animal facility. Two grams of beddings were taken from each cage of the same species and mixed together in a plastic bag. Two grams of the combined bedding were placed in a beaker and mixed with 40ml of saline solution. Ozone was fed into the beaker with a pipe from an ozone generator (Model FL-8A, Feili Electric Appliance Technology Co., Ltd, Shen Zhen, China) at a concentration of 500mg/h. 20μL of solution samples were subsequently taken from the beaker at time 0min, 30min, 45min and 60min after ozone treatment and diluted by 1000 times. 5μL was taken from the diluted sample and added to blood agar plates (Columbia blood agar base medium, diameter 9 CM, Bioivd Biotechnology (Zhengzhou) Co., Ltd). The plates (five per time point) were incubated for 48h.

2.2 Animal feces sampling and treatment with ozone

Feces from eight rabbits, which were kept in the same laboratory animal facility, were collected in a beaker and mixed. One gram of the mixture was placed into a beaker and mixed with 60ml of saline. Ozone was fed into the beaker at a concentration of 500mg/h, the treatment continued for 60min with samples taken at intervals of 0min, 15min, 30min, 45min and 60min. Solutions taken at the 0min point were diluted 10 times with saline before sampling. The sampling at each time point was achieved by transferring 5μL of the treated solution onto a blood agar plate for culture. Each time point had five plates.

The treatment and sampling procedures for dog feces were the same as that used for rabbits.

2.3 Culture count and bacteria identification

After 48h of culturing, the bacterial colonies in each plate (colony form unit, CFU/Plate) were counted and the types of the bacteria were classified with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MicroflexLT/SH. BRUKER, Germany).

2.4 Data analysis

Data of bacterial colony counts at different treatment time points were analyses using a related-samples Friedman two-way Analysis of Variance by Ranks (ANOVA) with post hoc pairwise comparisons (IBM SPSS Statistics v21). Proportions of Gram positive and Gram negative bacteria in samples before and after treatment were analysed using a 2x2 contingency table (Pearson Chi Square; IBM SPSS Statistics v21). Statistical significance for all tests was set at p<0.05.

3. Results

3.1 The disinfection efficacy of ozone on soiled beddings

Analysis shows that pathogen counts in samples taken from rat and mouse bedding at different time points during treatment with ozone are significantly different across all time points for each species (Friedman ANOVA: rat: F3, 30 = 74.95, p = 0.000; mouse: F3, 15 = 43.37, p = 0.000).

In rat bedding samples, post hoc pairwise comparisons against the untreated sample (time = 0min) reveal that ozone treatment to 30mins provides a significant reduction in bacterial colonies (F = 1.5, p = 0.000). Further treatment time does not produce significant reductions compared to preceding sampling time-points. However, treatment to 30, 45 and finally 60mins does provide additional benefits as reflected in greater effect size and an increasingly small rank mean and average pathogen count (see Table 1).

thumbnail
Download:
Table 1. The effectiveness of ozone treatment on soiled bedding of rats and mice.

https://doi.org/10.1371/journal.pone.0266223.t001

Data for mouse bedding treatment effect differ from those of the rat. In the mouse data only after 45min of treatment are bacterial colony counts significantly reduced compared to untreated samples (time = 0min) (F = 2.17, p = 0.000; see Table 1). As with rat bedding, treatment to 60min does result in the largest effect size (F = 2.833, p = 0.000) and also results in the lowest mean rank and average pathogen count of all sampling points.

As shown in Table 1, the ozone treatment of rat and mouse bedding for 30min killed 99% and 93% of pathogens in the beddings of the two species, respectively. Treatment for 60min killed over 99.70% of pathogens in both rat and mouse bedding.

3.2 The disinfection efficacy of ozone on animal feces

Analysis shows that pathogen counts in samples taken from feces of dogs and rabbits at different time points during treatment with ozone are significantly different across all time points (Friedman ANOVA: dog: F3, 15 = 58.61, p = 0.000; rabbit: F3, 15 = 55.36, p = 0.000).

In dog fecal samples, post hoc pairwise comparisons against the untreated sample (time = 0min) reveal that ozone treatment to 30min provides a significant reduction in bacterial colonies (F = 2.0, p = 0.005; Table 2). The effect size of the treatment continues to increase (also shown by progressive decrease in mean rank and average pathogen count) relative to time = 0min, up to time point t = 60mins (F = 6.697, p = 0.000; Table 2).

thumbnail
Download:
Table 2. The effectiveness of ozone treatment on the bacteria in dog and rabbit feces.

https://doi.org/10.1371/journal.pone.0266223.t002

Tests on rabbit feces data indicate that with ozone treatment a significant reduction in bacterial colony counts is only achieved at 30min (F = 3.811, p = 0.001; Table 2). The greatest effect of treatment is reached after 60min (F = 6.062, p = 0.000; Table 2).

As indicated in Table 2, ozone treatment of dogs and rabbits feces for 30min killed over 96% of pathogens for both species, and treatment for 60min killed nearly all of the pathogens in the feces of the both species (dog: 99.18%, rabbit: 99.9%).

3.3 Impact of ozone treatment on pathogen species in bedding

Table 3 shows the changes pathogen species profile in mouse and rat bedding before (time = 0min) and after (time = 60min) treatment with ozone. In rat bedding, before ozone treatment, there were six species of pathogen, of which five were Gram positive and one was Gram negative. After 60min of ozone treatment there was only one a Gram negative bacterium species (P. pasteurii) still present.

thumbnail
Download:
Table 3. The impact of ozone treatment on pathogens in the bedding of rats and mice.

https://doi.org/10.1371/journal.pone.0266223.t003

In the bedding of mice, there were eight species of pathogens (Gram positive = 4, Gram negative = 4) present before ozone treatment and only three bacteria species (Gram positive = 1, Gram negative = 2) persisted after treatment for 60min.

3.4 Impact of ozone treatment on pathogen species in feces

Table 4 illustrates the changes in pathogen species profile of the feces of rabbits and dogs before (time = 0min) and after (time = 60min) ozone treatment. In rabbit feces, there were two species of pathogens present before ozone treatment (both Gram positive) and only one of these was present after treatment. In dog feces, there were three bacterial species present before treatment (Gram positive = 2, Gram negative = 1) and two of these were still present after ozone treatment (Gram positive = 1, Gram negative = 1).

thumbnail
Download:
Table 4. The impact of ozone treatment on pathogens in the feces of rabbits and dogs.

https://doi.org/10.1371/journal.pone.0266223.t004

3.5 Sensitivity of bacteria to ozone

Table 5 shows that before ozone treatment, there were eight Gram negative pathogens and 11 Gram positive pathogens in the bedding of rats and mice and the feces of dogs and rabbits. After ozone treatment there were four Gram negative and three Gram positive pathogens present. There is no significant difference in sensitivity to ozone treatment between Gram negative and positive pathogens (χ21,26 = 0.465, p = 0.665).

thumbnail
Download:
Table 5. Comparison of sensitivity of Gram+ and Gram- bacteria to ozone treatment of the waste of rats and mice (bedding), and rabbits and dogs (feces).

https://doi.org/10.1371/journal.pone.0266223.t005

4. Discussion

In this study we found that there are pathogens in the soiled beddings of rat and mice and in the feces of dogs and rabbits. Ozone treatment can effectively kill these pathogens, and both Gram positive and negative pathogens are sensitive to this treatment.

The materials used as bedding have been found to hold a variety of bacteria, even when new [12], therefore they are now autoclaved before being used. Our current study confirms that various pathogens are present in the soiled bedding of specific pathogen free (SPF) as well as non-SPF facilities [13–19].

Domestic dogs may carry a range of pathogens [20, 21]. In dogs used in research, zoonotic and other pathogens, such as Salmonella spp., pathogenic dermal fungi, Brucella spp. and Rabies Virus that may cause serious illness and confound research, could be present and should be excluded according to the National Standards of China [20]. However, some pathogens that have only minor effects may still be present. In the current study, we found some pathogens that are not on the health-screening list of the National Standards. The situation is similar for rabbits.

The treatment of waste, such as bedding and feces, from laboratory animal facilities remains a significant challenge internationally [5, 6]. Incinerating and burying waste uses energy and contaminates the environment. A previously proposed and tested system for recycling bedding is impractical in most facilities due to space requirements for additional machinery [5, 6]. Using hydrogen peroxide vapour can inactivate pathogens in bedding, but this disinfection only works well against pathogens on the surface of waste. The current study demonstrates that treatment with ozone can effectively inactivate nearly all pathogens in the beddings, whether on the surface or in the core of the waste.

In China, the feces of research animals, including that of dogs and rabbits, is currently treated with agents containing chloride. Our study demonstrates that ozone treatment can effectively inactivate nearly all the pathogens in the feces of these animals. Unlike chloride agents which require special removal and treatment, ozone automatically decomposes in between 30 minutes and four hours, and thus is much less damaging to the environment.

As the ozone we used is generated from the air, it is nearly costless and it is certainly affordable. When using ozone to disinfect the beddings, a container is always used and the ozone is constricted in the container, thus will not contaminate the air.

Taken together, the ozone treatment of bedding and feces is an effective and environmentally friendly way to deal with the waste of animal facilities; saving energy and potentially enabling the reuse of waste as a fertilizer.

References

  1. 1. Allen PS, Lawrence J, Stasula U, Pallas BD, Freeman ZT. Effects of compressed paper bedding on mouse breeding performance and recognition of animal health concerns. J Am Assoc Lab Anim Sci. 2021 Jan 1;60(1):28–36. pmid:33323163
  2. 2. Bellin MA, Reitnauer KJ, Gomez AM, Lee-Stubbs RB, Ruff AL. Evaluation of a synthetic bedding substrate for mice (Mus musculus). J Am Assoc Lab Anim Sci. 2019 Mar 1;58(2):154–159. pmid:30803467
  3. 3. Pallas BD, Keys DM, Bradley MP, Vernasco-Price EJ, Sanders JD, Allen PS, et al. Compressed paper as an alternative to corn cob bedding in mouse (Mus musculus) cages. J Am Assoc Lab Anim Sci. 2020 Sep 1;59(5):496–502. pmid:32723424
  4. 4. Whiteside TE, Thigpen JE, Kissling GE, Grant MG, Forsythe D. Endotoxin, coliform, and dust levels in various types of rodent bedding. J Am Assoc Lab Anim Sci. 2010 Mar;49(2):184–9. pmid:20353693
  5. 5. Miyamoto T, Li Z, Kibushi T, Okano S, Yamasaki N, Kasai N. Utility of recycled bedding for laboratory rodents. J Am Assoc Lab Anim Sci. 2009 Jul;48(4):405–11. pmid:19653951
  6. 6. Miyamoto T, Li Z, Kibushi T, Yamasaki N, Kasai N. Use of soft hydrothermal processing to improve and recycle bedding for laboratory animals. Lab Anim. 2008 Oct;42(4):442–52 pmid:18782819
  7. 7. Baştürk E, Karataş M. Removal of pharmaceuticals by advanced treatment methods. J Environ Manage. 2021 Dec 15;300:113808. pmid:34649316
  8. 8. Criscuolo E, Diotti RA, Ferrarese R, Alippi C, Viscardi G, Signorelli C, et al. Fast inactivation of SARS-CoV-2 by UV-C and ozone exposure on different materials. Emerg Microbes Infect. 2021;10:206–210. pmid:33399524
  9. 9. Dubuis ME, Dumont-Leblond N, Laliberté C, Veillette M, Turgeon N, Jean J, et al. Ozone efficacy for the control of airborne viruses: Bacteriophage and norovirus models. PLoS One. 2020 Apr 10;15(4):e0231164. pmid:32275685
  10. 10. Martinelli M, Giovannangeli F, Rotunno S, Trombetta CM, Montomoli E. Water and air ozone treatment as an alternative sanitizing technology. J Prev Med Hyg. 2017 Mar;58(1):E48–E52. pmid:28515631
  11. 11. Sato H, Wananabe Y, Miyata H. Virucidal effect of ozone treatment of laboratory animal viruses. Jikken Dobutsu. 1990 Apr;39(2):223–9. pmid:2163330
  12. 12. Hogan JS, Smith KL. Bacteria counts in sawdust bedding. J Dairy Sci. 1997;80(8):1600–5. pmid:9276798
  13. 13. Bauer BA, Besch-Williford C, Livingston RS, Crim MJ, Riley LK, Myles MH. Influence of rack design and disease prevalence on detection of rodent pathogens in exhaust debris samples from individually ventilated caging systems. J Am Assoc Lab Anim Sci. 2016 Nov;55(6):782–788. pmid:27931317
  14. 14. Bohr URM, Selgrad M, Ochmann C, Backert S, König W, Fenske A, et al. Prevalence and spread of enterohepatic Helicobacter species in mice reared in a specific-pathogen-free animal facility. J Clin Microbiol. 2006 Mar;44(3):738–42. pmid:16517848
  15. 15. Hanson WH, Taylor K, Taylor DK. PCR testing of media placed in soiled bedding as a method for mouse colony health surveillance. J Am Assoc Lab Anim Sci. 2021 May 1;60(3):306–310. pmid:33952384
  16. 16. Kaliste E, Linnainmaa M, Meklin T, Torvinen E, Nevalainen A. The bedding of laboratory animals as a source of airborne contaminants. Lab Anim. 2004;38(1):25–37. pmid:14979985
  17. 17. Kim H, Bang J, Baek SH, Park JH. Eliminating murine norovirus, Helicobacter hepaticus, and intestinal protozoa by embryo transfer for an entire mouse barrier facility. Exp Anim. 2021 Aug 30. pmid:34456201
  18. 18. McInnes EF, Rasmussen L, Fung P, Auld AM, Alvarez L, Lawrence DA, et al. Prevalence of viral, bacterial and parasitological diseases in rats and mice used in research environments in Australasia over a 5-y period. Lab Anim (NY). 2011 Oct 20;40(11):341–50.
  19. 19. O’Connell KA, Tigyi GJ, Livingston RS, Johnson DL, Hamilton DJ. Evaluation of in-cage filter paper as a replacement for sentinel mice in the detection of murine pathogens. J Am Assoc Lab Anim Sci. 2021 Mar 1;60(2):160–167. pmid:33629939
  20. 20. Jimenez Castro PD, Venkatesan A, Redman E, Chen R, Malatesta A, Huff H, et al. Multiple drug resistance in hookworms infecting greyhound dogs in the USA. Int J Parasitol Drugs Drug Resist. 2021 Sep 2;17:107–117. pmid:34492564
  21. 21. Kim MW, Sharp CR, Boyd CJ, Twomey LN. A survey of enteric organisms detected by real-time PCR assay in faeces of dogs in Western Australia. Aust Vet J. 2021 Oct;99(10):419–422. pmid:34184250
  22. 22. Parrey AH, Sofi F, Ahmad M, Kuchay A. Aerococcus viridans infection presenting as cutaneous vasculitis in an immunocompetent patient. Reumatologia. 2016;54(6):318–320. pmid:28115783
  23. 23. Huang C. Extensively drug-resistant Alcaligenes faecalis infection. BMC Infectious Diseases, 2020, 20(1): 833. pmid:33176714
  24. 24. Garcia V, Abat C, Rolain J M. Report of the first Vagococcus lutrae human infection, Marseille, France. New Microbes New Infect, 2016, 9: 56–57. pmid:26862437
  25. 25. Altintas I, Andrews V, Larsen M V. First reported human bloodstream infection with Vagococcus lutrae. New Microbes New Infect, 2020, 34: 100649. pmid:32071726
  26. 26. Maraki S, Sarchianaki E, Barbagadakis S. Myroides odoratimimus soft tissue infection in an immunocompetent child following a pig bite: case report and literature review. Brazilian Journal of Infectious Diseases, 2012, 16(4): 390–392.
  27. 27. Benedetti P, Rassu M, Pavan G, Sefton A, Pellizzer G. Septic shock, pneumonia, and soft tissue infection due to Myroides odoratimimus: report of a case and review of Myroides infections. Infection, 2011, 39(2): 161–165. pmid:21246247
  28. 28. Kimouli M, Vrioni G, Papadopoulou M, Koumaki V, Petropoulou D, Gounaris A, Friedrich AW, Tsakris A. Two cases of severe sepsis caused by Bacillus pumilus in neonatal infants. International Journal of Medical Microbiology, 2012, 61(Pt 4): 596–599. pmid:22174371
  29. 29. Farhat H, Chachaty E, Antoun S, Nitenberg G, Zahar JR. [Two cases of Bacillus infection and immunodepression]. Medecine Et Maladies Infectieuses, 2008, 38(11): 612–614. pmid:18976873
  30. 30. Bentur HN, Dalzell AM, Riordan FAI. Central venous catheter infection with Bacillus pumilus in an immunocompetent child: a case report. Annals of clinical microbiology and antimicrobials, 2007, 6: 12. pmid:17967173
  31. 31. Tena D, Martinez-Torres JA, Perez-Pomata MT, Sáez-Nieto JA, Rubio V, Bisquert J. Cutaneous infection due to Bacillus pumilus: report of 3 cases. Clinical Infectious Diseases, 2007, 44(4): e40–42. pmid:17243047
  32. 32. Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clinical Microbiology Reviews, 2013, 26(4): 822–880. pmid:24092857
  33. 33. Gao W, Howden BP, Stinear TP. Evolution of virulence in Enterococcus faecium, a hospital-adapted opportunistic pathogen. Current Opinion in Microbiology, 2018, 41: 76–82. pmid:29227922