Ultraviolet Disinfection of Antibiotic Resistant Bacteria and Their

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Ultraviolet Disinfection of Antibiotic Resistant Bacteria (ARBs) and their Antibiotic Resistance Genes (ARGs) in Water and Wastewater Chad W McKinney, and Amy Pruden Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es303652q • Publication Date (Web): 15 Nov 2012 Downloaded from http://pubs.acs.org on November 28, 2012

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Environmental Science & Technology

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Ultraviolet Disinfection of Antibiotic Resistant

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Bacteria (ARBs) and their Antibiotic Resistance

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Genes (ARGs) in Water and Wastewater

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AUTHOR NAMES. Chad W. McKinney1 & Amy Pruden1*

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RECEIVED DATE

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AUTHOR ADDRESS. 1Via Department of Civil and Environmental Engineering, Virginia Tech,

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Blacksburg, VA 24061

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*CORRESPONDING AUTHOR. Via Department of Civil and Environmental Engineering, Virginia

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Tech, Blacksburg, VA 24061. Email: [email protected]; Phone: (540) 231-3980; Fax: (540) 231-7916

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KEYWORDS. UV, antibiotic resistance genes, MRSA, vancomycin-resistant Enterococci,

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Pseudomonas aeruginosa, Escherichia coli, wastewater effluent

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ABSTRACT. Disinfection of wastewater treatment plant effluent may be an important barrier for

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limiting the spread of antibiotic resistant bacteria (ARBs) and antibiotic resistance genes (ARGs).

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While ideally disinfection should destroy ARGs, in order to prevent horizontal gene transfer to

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downstream bacteria, little is known about the effect of conventional water disinfection technologies on

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ARGs. This study examined the potential of UV disinfection to damage four ARGs; mec(A), van(A),

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tet(A), and amp(C), both in extracellular form and present within a host ARBs: methicillin-resistant ACS Paragon Plus Environment

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Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), Escherichia coli

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SMS-3-5, and Pseudomonas aeruginosa 01, respectively. An extended amplicon-length quantitative

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polymerase chain reaction assay was developed to enhance capture of ARG damage events and also to

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normalize to an equivalent length of target DNA (~1000 bp) for comparison. It was found that the two

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Gram positive ARBs (MRSA and VRE) were more resistant to UV disinfection than the two Gram

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negative ARBs (E. coli and P. aeruginosa). The two Gram positive organisms also possessed smaller

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total genome sizes, which could also have reduced their susceptibility to UV because of fewer potential

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pyrimidine dimer targets. An effect of cell type on damage to ARGs was only observed in VRE and P.

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aeruginosa, the latter potentially because of extracellular polymeric substances. In general, damage of

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ARGs required much greater UV doses (200 – 400 mJ/cm2 for 3- to 4-log reduction) than ARB

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inactivation (10-20 mJ/cm2 for 4- to 5-log reduction). The proportion of amplifiable ARGs following

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UV treatment exhibited a strong negative correlation with the number of adjacent thymines (Pearson r

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< -0.9; p < 0.0001). ARBs surviving UV treatment were negatively correlated with total genome size

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(Pearson r< -0.9; p MRSA > E. coli SMS-3-5 > P. aeruginosa 01 (Figure 2). ACS Paragon Plus Environment

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Variable Responses of ARGs to UV Based on pooling of each individual ARG C/Co measurement across intracellular and extracellular data sets and aqueous matrix type at each UV dose (25, 50, 100, and 200 mJ/cm2), the order of least susceptible to most susceptible to treatment was: ampC ≥ tet(A) > vanA ≥ mecA (Figure 3). This hierarchy was consistent with the correlation analysis in which the ARG with the least potential thymine dimer sites (ampC) incurred the least UV damage, while mecA carried the most potential thymine dimer sites and was the most readily damaged. It was noted that ampC and tet(A) were not significantly different at UV doses of 25 (p = 0.95), 50 (p = 0.82), 100 (p = 0.72), or 200 mJ/cm2 (p = 0.39), but ampC was significantly higher than tet(A) after 300 (p = 0.066) and 400 mJ/cm2 (p = 0.057) treatments. Nonetheless, ampC and tet(A) were consistently more recalcitrant than van(A) and mec(A) (p ≤ 0.067) (Figure 3). vanA was also significantly more resistant to UV than mecA at all doses (p ≤ 0.045), except 25 (p = 0.69) and 50 mJ/cm2 (p = 0.18).

DISCUSSION This study provided a controlled examination of the potential for UV to disinfect four key antibiotic-resistant pathogens of concern. P. aeruginosa pose a major challenge when they colonize hospital taps [32], and multiple-antibiotic resistant strains are blamed for bacteremia in burn victims, urinary-tract infections in catheterized patients, nosocomial pneumonia in patients with respirators, and are the primary cause of morbidity and mortality in cystic fibrosis patients [19]. E. coli are fecal organisms and are well-known to survive wastewater treatment. Virulent strains of E. coli are one of the major causes of gastroenteritis, urinary tract infections, and neonatal meningitis. Enterococci are also fecal-borne organisms and VRE in particular are of critical interest because vancomycin is an antibiotic of last resort. vanA ARGs have been found in drinking water biofilms [33], which has brought to light the fact that ARGs can persist much further in the water supply than might have been expected. Finally, MRSA, which are responsible for about 10,800 deaths per year in the U.S. [34], ACS Paragon Plus Environment

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have recently been detected in the effluent of four U.S. wastewater treatment plants [35]. Thus, there is the potential for all four ARBs to be present in treated water or wastewater, and although broader surveys of their occurrence are needed, this study provides information on the feasibility of UV treatment under worst-case scenario conditions (i.e. water and wastewater containing high concentrations of pathogenic bacteria that are resistant to multiple and/or last resort antibiotics). While UV inactivation of these bacteria has been investigated previously, prior research has not compared the four targets together under conditions representative of water or wastewater. Also, the ARB inactivation results provided an important baseline for comparison of the relative effects of UV on intracellular versus extracellular ARGs. All four ARBs were readily inactivated by UV treatment and 5-log removal was achieved by doses between 10 and 20 mJ/cm2. However, each individual ARB was characterized by a unique disinfection curve, with the two Gram positive ARBs (VRE and MRSA) being more difficult to inactivate than the two Gram negative ARBs (E. coli and P. aeruginosa). This suggests that the thicker peptidoglycan layer characteristic of Gram positive organisms may offer some protection against UV inactivation. Sheldon and colleagues (2005) [36] similarly observed MRSA to be more resistant to UV than P. aeruginosa when applied to eliminate the organisms on human skin. Interestingly, they also observed that P. aeruginosa was adept at photoreactivation, while MRSA was not. This is a possible explanation for the persistence of ampC genes present within P. aeruginosa cells in this study, even at very high UV doses. Genome size appeared to play a role in ARB inactivation and including P. aeruginosa in the study provided representation of a bacterium with an extremely large genome [37]. The fact that a negative correlation between genome size and ARB inactivation was found suggests that larger genomes inherently possess more targets for UV damage and are therefore more susceptible. However, this study suggests that direct damage of DNA as a result of pyrimidine dimer formation is not the only mechanism of UV inactivation. ARB inactivation actually decreased with increased adjacent thymine ACS Paragon Plus Environment

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sites per genome, but increased with adjacent cytosines, which was opposite to the trend observed for ARGs. Together with the fact that ARG damage required a much greater UV dose than bacterial inactivation, suggests more complex cellular responses to UV light resulting in inactivation. The differences between Gram positives and Gram negatives also indicate a potential role of the cell envelope. Of particular interest in this study was the potential for UV to impart damage to ARGs in order to reduce risk of them being transformed to new downstream hosts. qPCR was selected to assay DNA damage because DNA damage (e.g., oxidized bases, abasic sites, and thymine dimers) interferes with amplification by Taq polymerase [38-40]. However, qPCR is typically designed for amplicon lengths of only 75-100 bp, which limits the potential to capture DNA damage. In order to maximize the capability to detect DNA damage by qPCR, the assays were adapted for long target amplification. Suess and Colleagues (2009) [40] recently investigated amplicon lengths of 100, 500, and 900 bp and observed that DNA damage detection improves as amplicon size is increased. In the present study, it was possible to further extend the length of the target to ~1,000 bp through adaptation of recently patented EvaGreen® chemistry (Bio-Rad), which is reported by the manufacturer to be less inhibitory to PCR than SybrGreen [41]. Also, the Sso7d-fusion polymerase in the Supermix is reported to stabilize the polymerase:template complex, increasing processivity and speed of amplification [42]. The near-equivalent length of the target amplicons also normalized this variable and enabled comparison of the effects of UV among ARGs. While qPCR is not a direct assay of loss of DNA function, several studies have demonstrated that UV-damaged genes, including ARGs, can no longer function within a host bacterial cell [6]. This study revealed that damage to ARGs requires at least an order of magnitude higher UV doses than does inactivation of host bacterial cells. 3- to 4-log damage to ARGs required doses ranging from 200-400 mJ/cm2. Notably, the ampC and tet(A) ARGs were significantly more recalcitrant than the mecA and vanA ARGs. DNA sequence characteristics appeared to be the primary driver of the ACS Paragon Plus Environment

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differences observed among ARGs. Strong correlations were observed between the number of adjacent thymines and inactivation, with mecA and vanA possessing more adjacent thymines. The cell envelope appeared to also play some protective role for ARGs in one Gram positive (VRE/vanA) and one Gram negative (P. aeruginosa/ampC) ARB (Figure 3). ampC present within P. aeruginosa was the most resistant to UV, which may be a result of the excessive extracellular polymeric substances produced by this ARB, which was readily apparent when plating these cells. Also, as noted above, P. aeruginosa are known to be adept at photorepair [36]. While the fact that aqueous matrix had little impact on disinfection in this study is intuitive, it was still important to confirm that this was the case in order to support interpretation of the results in the light of variable wastewater compositions. The aqueous matrix did not have a dramatic effect on either ARB inactivation or ARG damage, suggesting that the results of this study may be broadly applicable to a variety of waters. However, a note of caution is still warranted that effectiveness of UV treatment will be hampered by the presence of turbidity, which was filtered out in this study. In this study, VRE was the only ARB that consistently responded differently in the PBS versus wastewater effluent media. It is possible that different cells interact differently with dissolved constituents in the aqueous matrix, which in turn affects their susceptibility to disinfection. Sheldon and colleagues (2005) [36] similarly observed that salt concentration impacted the efficacy of UV treatment against MRSA. The U.S. Environmental Protection Agency Ultraviolet Disinfection Guidance Manual for the Final Long Term 2 Enhanced Surface Water Treatment Rule provides federal guidelines for UV doses required to kill/inactivate viruses, Giardia, and Cryptosporidium. Direct guidelines are not provided for bacteria, because they are generally easier to kill than these model microorganisms [43]. According to the guidelines, the highest recommended UV dose of 186 mJ/cm2 results in 4-log removal and/or inactivation of viruses [21]. This UV dose requirement will likely effectively inactivate all four target

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ARB investigated in this study, and damage mecA and vanA by 3- to 4-log and tet(A) and ampC by 1to 2-log. This study suggests that UV disinfection holds only limited potential to damage ARGs in water and wastewater effluents. The levels required to achieve 3- to 4-log reduction in ARGs would be impractical for water utilities to impose, and also the potential for photoreactivation would be greater under field conditions, whereas this study aimed to limit this factor. There is a need to similarly assess the potential of other water disinfection technologies, such as chlorination, chloramination, or ozone, to limit the spread of ARGs. UV combined with a hydroxyl-producing catalyst may offer more extensive ARG damage than observed in this study. For example, bactericidal activity of UV was recently reported to be greatly enhanced by a nano-TiO2/Ag catalyst [44].

ACKNOWLEDGEMENTS Funding for this research was provided by the Virginia Tech Institute for Critical Technology and Applied Science TSTS Award 11-26 and the National Science Foundation CBET CAREER award # 0547342. The findings do not represent the views of the funding sponsors.The authors would also like to thank Brittany Ott and Robert Miles Ellenberg for their laboratory assistance.

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Oliveira, D. C.; de Lencastre, H., Multiplex PCR strategy for rapid identification of structural

types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 2002, 46, (7), 2155-2161. 49.

Dalsgaard, A.; Forslund, A.; Sandvang, D.; Arntzen, L.; Keddy, K., Vibrio cholerae O1

outbreak isolates in Mozambique and South Africa in 1998 are multiple-drug resistant, contain the SXT element and the aadA2 gene located on class 1 integrons. J. Antimicrob. Chemother. 2001, 48, (6), 827-838. 50.

Ng, L. K.; Martin, I.; Alfa, M.; Mulvey, M., Multiplex PCR for the detection of tetracycline

resistant genes. Mol. Cell. Probes 2001, 15, (4), 209-215. 51.

Clark, N. C.; Cooksey, R. C.; Hill, B. C.; Swenson, J. M.; Tenover, F. C., Characterization of

glycopeptide-resistant Enterococci from United-States Hospitals. Antimicrob. Agents Chemother. 1993, 37, (11), 2311-2317.


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TABLES.

Table 1: Primers used in this study Primer ampC-FP ampC-RP mecA-FP mecA-RP tet(A)-FP tet(A)-RP vanA-FP vanA-RP

TA

Amplicon Length

70.9 °C

1006 bp

51.7 °C

1018 bp

57.2 °C

1054 bp

55.7 °C

1030 bp

Sequence Reference 5' CGG CTC GGT GAG CAA GAC CTT C [45] 5' GAA GCG CTC ATG GCA CCA TCA TAG CC [46] 5' CGC AAC GTT CAA TTT AAT TTT GTT AA [47] 5' CCA CTT CAT ATC TTG TAA CG [48] 5' GTA ATT CTG AGC ACT GTC GC [49] 5' CAT AGA TCG CCG TGA AGA GG [50] 5' CAT GAA TAG AAT AAA AGT TGC AAT A [51] 5' CCC CTT TAA CGC TAA TAC GAT CAA

TA = annealing temperature, FP = forward primer, RP = reverse primer

[45-51] Table 2: Pyrimidine dimer counts for ARGs and ARB ARG

Amplicon Length (bp)

mecA vanA tet(A) ampC

1018 1030 1054 1006

ARB

Genome Size (bp)

MRSA VRE E. coli SMS-3-5 P. aeruginosa 01

2,872,769 2,826,716 5,068,389 6,264,404

TT 263 190 81 40 TT 706,446 638,915 746,734 376,612

Dimers (Count) CC TC CT 59 114 100 101 115 105 184 135 102 206 106 129 Dimers (Count) CC TC CT 145,879 302,081 277,579 199,341 373,437 318,366 589,024 584,759 517,931 1,171,802 766,117 707,725

Total 536 511 502 481 Total 1,431,985 1,530,059 2,438,448 3,022,256

Dimers/Amplicon Length (%) TT CC TC CT Total 25.8 5.80 11.2 9.82 52.7 18.4 9.81 11.2 10.2 49.6 7.69 17.5 12.8 9.68 47.6 3.98 20.5 10.5 12.8 47.8 Dimers/Genome Size (%) TT CC TC CT Total 24.6 5.08 10.5 9.66 49.8 22.6 7.05 13.2 11.3 54.1 14.7 11.6 11.5 10.2 48.1 6.01 18.7 12.2 11.3 48.2

Dimers/Total Dimers (%) TT CC TC CT 49.1 11.0 21.3 18.7 37.2 19.8 22.5 20.5 16.1 36.7 26.9 20.3 8.32 42.8 22.0 26.8 Dimers/Total Dimers (%) TT CC TC CT 49.3 10.2 21.1 19.4 41.8 13.0 24.4 20.8 30.6 24.2 24.0 21.2 12.5 38.8 25.3 23.4

TT = thymine Dimer, CC = cytosine dimer, TC = 5’-thymine-cytosine dimer, CT = 5’-cytosine-thymine dimer MRSA = methicillin-resistant Staphylococcus aureus, VRE = vancomycin-resistant Enterococci


25


1 2 578 3 4 579 5 6 7 580 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 581 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

FIGURES.

Figure 1: Representative standard curve for a qPCR run with a 1,000 bp amplicon. Cq = Threshold cycle; E = efficiency; SYBR = program name (EvaGreen® was used as intercalating dye).


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Figure 2: Ultraviolet (UV, λ = 254 nm) disinfection curves of antibiotic resistant bacteria (ARBs). Error bars are standard error of the mean. MRSA = Methicillin-resistant Staphylococcus aureus. VRE = Vancomycin-resistant Enterococcus faecium. PBS = Phosphate buffered saline solution. WW = Filtered wastewater.


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100 mecA-EXC mecA-INC vanA-EXC vanA-INC tet(A)-EXC tet(A)-INC ampC-EXC ampC-INC

10-1 ARG C/C0

10-2 100

10-3

10-1 ARG C/C0

1 2 584 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 28

10-2

10-3

10-4

10-4 400

0

500 600 700 800 900 UV254 Fluence Dose (mJ/cm2)

1000

50 100 150 200 250 300 350 400 UV254 Fluence Dose (mJ/cm2)

Figure 3: Ultraviolet (UV, λ = 254 nm) disinfection curves of antibiotic resistant genes (ARGs). Error bars are standard error of the mean. mecA = Methicillin resistance gene. vanA = Vancomycin resistance gene. tet(A) = Tetracycline resistance gene. ampC = Ampicillin resistance genes. INC = Intracellular. EXC = Extracellular. Insert: ampC-INC disinfection curve from 400 to 1000 mJ/cm2.


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Ultraviolet Disinfection of Antibiotic Resistant Bacteria and Their

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