In order to investigate the effect of UV disinfection on the inactivation of bacteria in water and the effect of drug resistance, this study used three species of E. coli with different drug resistance for UV disinfection experiments, please read on for details.
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UV disinfection is one of the most widely used wastewater disinfection technologies, which has the advantages of high sterilization efficiency, no disinfection by-products, effective killing of Cryptosporidium and Giardia, and low disinfection effect affected by water temperature and pH. In recent years, with the concern of bacterial resistance and the in-depth research of UV disinfection technology, it is gradually found that the effect of UV disinfection on different bacteria is different. For example, Gram-positive bacteria are more resistant to UV than Gram-negative bacteria, and Kim et al. found that the proportion of tetracycline-resistant bacteria increased after UV disinfection, and Guo et al. further demonstrated that UV disinfection is bioselective, leading to an enrichment of heterotrophic bacteria resistant to sulfonamide, vancomycin, rifampicin, tetracycline, and chloramphenicol in the effluent of the diphtheria tank, while the proportion of heterotrophic bacteria resistant to cephalexin, erythromycin, gentamicin, and ciprofloxacin decreased. The proportion of heterotrophic bacteria resistant to the drugs was reduced.
Most of these studies illustrated the effect of UV disinfection on bacterial resistance by examining the changes in the resistance rates of a large group of bacteria to different antibiotics before and after UV disinfection. The changes in the concentration and resistance of specific bacteria before and after UV disinfection have rarely been reported. In this study, three species of E. coli with different drug resistance were selected and their inactivation and drug resistance were investigated by UV disinfection experiments with different UV irradiation doses in order to understand the mechanism of UV disinfection on bacteria more deeply and provide a reference for the rational use of wastewater disinfection technology.
1 Experimental method
1)Experimental strains and sample preparation
E. coli strain ATCC25922 and E. coli resistant strains SER2 and SER6 isolated from secondary effluent of a municipal wastewater treatment plant were selected as the test strains. The resistance status of these strains to nine common antibiotics, namely tetracycline (TET), sulfamethoxazole (SMZ), ampicillin (AMP), streptomycin (STP), cefotaxime (CTX), gentamicin (GEN), chloramphenicol (CHL), ciprofloxacin (CIP) and norfloxacin (NOR), is shown in Table 1.
Single colonies of the strains were picked in 5 mL of liquid LB medium and incubated overnight at 37 °C. The samples were centrifuged at 4 °C for 10 min at 10 000 r/min, washed twice with 0. 1 mol/L phosphate buffer solution (PBS, pH 7. 4), and transferred to 400 mL of 0. 1 mol/L PBS to make E. coli samples with a concentration of about 106 CFU/mL.
Table 1 Resistance phenotypes of the experimental strains to nine common antibiotics
| Antibiotics | TET | SMZ | AMP | GEN | STP | CTX | CHL | CIP | NOR |
|---|---|---|---|---|---|---|---|---|---|
| ATCC25922 | – | – | – | – | – | – | – | – | – |
| SER2 | + | + | – | – | – | – | – | – | – |
| SER6 | + | + | + | + | + | + | – | – | – |
Note: Testing was performed using the American Clinical and Laboratory Standards Institute (CLSI) standards. ” -” indicates that the strain is antibiotic sensitive, and” + “indicates that the strain is antibiotic-resistant.
2)UV disinfection test
A 40 mL sample was taken in a 90 mm diameter sterilized Petri dish and placed in a UV parallel beam meter to receive UV irradiation. The samples were stirred magnetically to ensure the uniformity of E. coli distribution. The UV intensity of 100 μW/cm² was measured by a UV intensity meter (UV-B type, Beijing Normal University Optoelectronics Instrument Factory). A series of different UV irradiation doses were obtained by controlling the irradiation time.
Three UV disinfection experiments were performed for each strain. E. coli concentrations were determined by nutrient agar plate counting of samples before and after UV disinfection. The logarithmic inactivation rate (R) at each UV irradiation dose was :
R = log(N0/Ni)
Where: N0 is the concentration of E. coli (CFU/mL) in the sample without UV disinfection (original); Ni is the concentration of E. coli (CFU/mL) in the sample after UV disinfection at a certain dose.
3)Drug sensitization assay
The drug sensitivity assay was performed by the K-B (Kirby-Bauer) paper agar diffusion method. The original samples containing E. coli strain SER6 were sterilized by UV light at 80 mJ/cm², and a number of single colonies were randomly selected and made into a suspension with 0. 5 Mackay turbidity in sterile 0. 9% NaCl solution. The samples were incubated at 35℃ for 18~24 h. The diameter of the inhibition ring was measured by vernier calipers, and the sensitivity of the tested strains to antibiotics was determined according to the CLSI enterobacterial drug sensitivity test.
4) Scanning electron microscopy (SEM) observation
The original samples containing E. coli drug-resistant strain SER6 and the samples after UV disinfection at 200 mJ/cm² were taken separately, and the aqueous samples were filtered with suspension solution using 0. 22 μm microporous membrane, eluted with 3% beef paste, centrifuged, and placed in 4% para formaldehyde overnight at 4 °C. The supernatant was poured out and washed twice with pure water. The ethanol-isoamyl acetate solution (v/v, 1:1) was used with 30%, 50%, 70%, 80%, 90%, 95%, 100% ethanol. The isoamyl acetate solution was washed sequentially, in which 90%, 95%, 100% ethanol, and pure isoamyl acetate were washed twice for 15 min each, and the rest of the concentrations were washed once for 10 min. natural drying for 1 week, gold spraying for 120 s, and SEM (Japan, JSM-6700F) photography for observation.
5) Statistical analysis of data
The experimental data were analyzed using the PASW Statistics 18. 0 data analysis software. The t-test in the PASW Statistics 18.0 data analysis software to test whether the survival of E. coli with different drug resistance was significant after disinfection with a certain dose of UV radiation. The survival rate of E. coli with different drug resistance after disinfection with a given dose of UV radiation was tested for significance, and whether there was a significant difference in the mean diameter of the inhibition ring of strain SER6 before and after UV disinfection. The mean diameter of SER6 was tested for significant differences.
2 Results and Discussion
1)Effect of UV light on inactivation of experimental strains
The log inactivation rates of the three experimental strains increased with the increase of UV irradiation dose and increased very rapidly in the range of 0-20 mJ/cm², and then gradually slowed down. At a dose of 20 mJ/cm², the mean log inactivation rates of E. coli ATCC25922, SER2, and SER6 were 4. 75, 5. 04 and 3. 83, respectively; while at a dose of 80 mJ/cm², the log inactivation rates of the three strains reached 5. 51, 5. 68 and 5. 29 (Figure 1). Thus, although the inactivation curves of these three E. coli species by UV disinfection were different, all of them could produce good inactivation effects at high irradiation doses.
Fig. 1 Inactivation of tested strains by ultraviolet disinfection
The inactivation curves of E. coli strains ATCC25922 and SER2 were relatively similar for UV disinfection. The logarithmic inactivation rates of these two strains were not significantly different at most irradiation doses (p > 0. 05). The inactivation curves of strain SER6 were more different from those of ATCC25922, and the log inactivation rates of SER6 were lower than those of ATCC25922 at the same UV dose. t-test demonstrated that the log inactivation rates of SER6 and ATCC25922 were significantly different in the range of 0-20 mJ/cm² (p < 0. 05), while the log inactivation rates of SER6 and ATCC25922 were significantly different in the range of 40 mJ/cm² and 80 mJ/cm² (p < 0. 05). There was no significant difference between SER6 and ATCC25922 at 40 mJ/cm² and 80 mJ/cm² (p > 0.05).
This result indicates that the response of dioresistant and antibiotic-sensitive bacteria to UV disinfection is very similar, and the killing effect of antibiotic-sensitive bacteria can be used to estimate the inactivation of dioresistant bacteria. However, in order to meet the domestic primary A standard for wastewater discharge, the design of UV disinfection systems for wastewater plants (GB/T 19839-2005) requires that the UV irradiation dose should not be less than 20 mJ/cm², while multi-drug resistant bacteria are more resistant to UV disinfection, and lower irradiation doses have a very limited effect on their inactivation. For example, at 10 mJ/cm², the log inactivation rate of SER6 was only 2.42. The poor inactivation of highly resistant multi-drug resistant strains by low dose UV disinfection is likely to be responsible for the increase in resistance rate after UV disinfection of secondary treatment effluent from wastewater plants. For example, Meckes et al. found that coliform resistance to tetracycline and chloramphenicol was higher after UV disinfection at 45 mJ/cm² than before disinfection and that the proportion of multi-drug resistant coliforms was significantly higher.
Some previous studies have suggested that drug-resistant E. coli are as resistant to UV as antibiotic-sensitive bacteria, but the drug-resistant E. coli used were not multi-drug-resistant strains, so the results obtained were one-sided. In this study, we found that multi-drug resistant E. coli were more resistant to UV light, and it is speculated that the reason for this may be related to the presence of plasmids containing multi-drug resistance genes in multi-drug resistant E. coli, which increase the resistance of bacteria to UV light while making them resistant to drugs.
2) Effect of UV disinfection on drug resistance of experimental strains
The diameter of the inhibition ring produced by the drug sensitivity assay of the strain indicates its sensitivity to the antibiotic. Several experiments were conducted using E. coli SER6, and the distribution of inhibition ring diameters produced by the nine antibiotics is shown in Table 2. Before UV disinfection experiments, SER6 was resistant to TET, SMZ, AMP, GEN, STP and CTX, sensitive to CHL and NOR, and sensitive or intermediate to CIP; after UV disinfection experiments, its susceptibility to these antibiotics remained largely unchanged.
If we analyze the diameter values of the inhibition rings, we find that the diameter of the inhibition rings of TET, SMZ, AMP, and STP is very stable before and after the UV disinfection experiments, and even the coefficient of variation of many experiments is 0; while the diameter of the inhibition rings produced by GEN, CHL and NOR actually changed. The ring diameter of GEN became smaller after UV disinfection and was significantly different from that before the disinfection experiment (p < 0. 05); the ring diameter of CHL also became smaller (p < 0. 05); while the ring diameter of NOR became larger (p < 0. 05). This result suggests that the stability of resistance to different antibiotics differs somewhat, even for multi-drug resistant strains. UV irradiation, as an environmental factor, has a mutagenic effect on strains. Those bacteria that are not stably resistant may be altered by this mutagenic effect, which may be manifested by some individual bacteria, as visualized in the results of the drug sensitivity assay, where the distribution of inhibition ring diameters became discrete and even showed significant changes in the mean diameter values.
Table 2 Diameter of inhibition zone related to E. coli SER6 before and after ultraviolet irradiation
| Drug sensitive paper sheets | Diameter range before UV disinfection (mm) | Average value before UV disinfection(mm) | Coefficient of variation before UV disinfection (% ) | Diameter range after UV disinfection (mm) | Average value after UV disinfection(mm) | Coefficient of variation after UV disinfection (% ) |
|---|---|---|---|---|---|---|
| TET | 6.0 | 6.0 | 0 | 6.0 | 6.0 | 0 |
| SMZ | 6.0 | 6.0 | 0 | 6.0 | 6.0 | 0 |
| AMP | 6.0 | 6.0 | 0 | 6.0 | 6.0 | 0 |
| GEN | 8.0 ～ 8.8 | 8.3 | 3.5 | 6.5~8.5 | 7.4 | 9.5 |
| STP | 6.0 | 6.0 | 0 | 6.0 | 6.0 | 0 |
| CTX | 9.0~10.0 | 9.6 | 3.5 | 7.5~10.8 | 9.3 | 10.6 |
| CHL | 21.5~23.5 | 22.3 | 4.9 | 14.0~19.5 | 17.0 | 8.7 |
| CIP | 19.5~21.3 | 20.4 | 3.3 | 18.0~21.0 | 20.1 | 5.4 |
| NOR | 17.5~18.5 | 18.1 | 2.2 | 17.5~23.5 | 21.6 | 9.2 |
3) Effect of UV disinfection on the cytoarchitecture of the bacterium
The surface and overall structure of the bacteriophage could be clearly seen by SEM by magnifying the SER6 bacteriophage 15 000 times (Figure 2). Comparing the condition of the bacteriophage before and after the UV disinfection experiment, it was found that after 20 mJ/cm² UV disinfection, the bacteriophage in the sample was intact with a smooth surface, and there was no obvious difference with the bacteriophage of the original sample; however, after 80 mJ/cm² UV disinfection, the surface of individual bacteriophage showed depression; after 200 mJ/cm² UV disinfection, many bacteriophages showed surface After disinfection at 200 mJ/cm2, the surface of many organisms was depressed or wrinkled, and some organisms were even broken and the intracellular material flowed out.
The cell wall of bacteria not only maintains the intrinsic morphology of bacteria but also plays an important role in the exchange of substances between the cell and the external environment. The complex structure of the outer membrane layer of the cell wall of Gram-negative bacteria, which can effectively block the entry of antibiotics and other substances into the bacterium, is one of the natural drug resistance mechanisms of certain bacteria. Damage to the surface of bacteria implies a weakening or loss of resistance to external adverse factors, and their drug resistance characteristics and survival ability are inevitably altered. Most of the previous studies have concluded that the inactivation of bacteria by UV light is mainly due to the inactivation of bacteria.
The results of this experiment directly demonstrate that UV disinfection causes damage to the surface and integrity of the bacteria from a microscopic point of view. In addition to the high dose of UV irradiation, this phenomenon is also closely related to the wavelength of UV light: it has been reported that after 1 h of irradiation with a 500 W UV lamp (primary wavelength 365 nm), the surface of the bacterium was damaged, but the structural integrity was not changed. The UV source used in this study was a 20 W low-pressure UV lamp with a main wavelength of 253.7 nm, which is more powerful in killing microorganisms and more likely to cause serious damage to the morphology of the bacterium.
3 Conclusion
(1) The log inactivation rates of the three experimental strains of E. coli ATCC25922, SER2, and SER6 increased with increasing UV irradiation dose, especially in the range of 0 to 20 mJ/cm². 200 mJ/cm² high dose UV irradiation caused damage to the bacterial cell wall and even destroyed the integrity of the bacterial body.
(2) The UV disinfection inactivation curves of E. coli ATCC25922 and SER2 were similar, while E. coli SER6 was more resistant to UV disinfection, and its logarithmic inactivation rate was significantly lower in the range of 0-20 mJ/cm² irradiation dose.
(3) For E. coli SER6 strains, the diameter of the inhibition rings produced by gentamicin, chloramphenicol, and norfloxacin were significantly different before and after UV disinfection at 80 mJ/cm², but the resistance phenotype did not show significant changes. There was no significant change in the resistance phenotype.
References
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