Inactivation and Gene Expression of a Virulent Wastewater

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Inactivation of a virulent wastewater Escherichia coli and non-virulent commensal Escherichia coli DSM1103 strains and their gene expression upon solar irradiation Nada Al-Jassim, David Mantilla-Calderon, Tiannyu Wang, and Peiying Hong Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05377 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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Environmental Science & Technology

Graphical abstract 294x156mm (150 x 150 DPI)

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Inactivation and gene expression of a virulent wastewater Escherichia coli strain and the

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non-virulent commensal Escherichia coli DSM1103 strain upon solar irradiation

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Nada Al-Jassim 1, David Mantilla-Calderon 1, Tiannyu Wang 1, Pei-Ying Hong * 1

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1. King Abdullah University of Science and Technology (KAUST), Water Desalination and Reuse Center (WDRC), Biological and Environmental Sciences & Engineering Division (BESE), Thuwal, 23955-6900, Saudi Arabia

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* Corresponding author: Pei-Ying Hong Email: [email protected] Phone: +966-12-8082218

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Keywords: ultraviolet disinfection; decay kinetics; antibiotic-resistant bacteria; New Delhi metallo-beta-lactamase; virulence; transcriptomics

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Running title: Decay kinetics and gene expression profiles of E. coli strains upon solar irradiation

1 ACS Paragon Plus Environment

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Abstract

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This study examined the decay kinetics and molecular responses of two E. coli strains upon solar

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irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated

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from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E.

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coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer

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solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared

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with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs. 1.33 ± 0.52 h and 2.04 ± 0.36

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min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar

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irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in
12 h of sunlight irradiation (Figure 1A and C). In 9 ACS Paragon Plus Environment

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contrast, E. coli DSM1103 exhibited a significantly shorter lag phase of 1.33 ± 0.52 h (p
0.05). E. coli PI-7 exhibited mean lag phase lengths of 6.64 ± 0.48 h and 6.30 ± 1.18 h in

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effluent and chlorinated effluent, respectively (Figure 2A and B). E. coli DSM1103 exhibited

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mean lag phase lengths of 1.29 ± 0.49 h and 0.71 ± 0.76 h in effluent and chlorinated effluent,

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respectively (Figure 2C and D). After the lag phase, E. coli PI-7 exhibited a mean half-life of

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4.36 ± 1.13 min and 5.23 ± 1.40 min in effluent and chlorinated effluent, respectively (Table S6).

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The half-life of E. coli PI-7 in chlorinated effluent was significantly longer than the half-life

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measured for E. coli PI-7 in buffer (p = 0.0167). For E. coli DSM1103, the half-life value in

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effluent was 4.03 ± 0.36, and the half-life was 2.05 ± 0.16 min in chlorinated effluent (Table S6).

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The half-life in the effluent was significantly longer than that in the buffer or the chlorinated

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effluent (p = 0.0012).



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3.2. Extent of differential gene expression response throughout the different phases of solar

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irradiation

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Prior to transcriptomic analysis, general genomic comparisons were performed to provide

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insight into the differences between the two isolates’ genomes. The genome of E. coli DSM1103

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(5.2 Mb) was found to be larger than the available genome of PI-7 (4.73 Mb) (Table S1). Based

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on the currently available mapped genomes, E. coli DSM1103 exhibited a larger total number of

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annotated genes than PI-7 (4,808 vs. 4,471, respectively) as well as more unique gene sequences

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and annotated gene functions (910 vs. 489 and 185 vs. 155, respectively). The annotated

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functions were further separated by functional category (Table S7) to illustrate the shared and

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unique genes between the two bacterial strains.

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RNA-seq was performed to examine the molecular responses of the two isolates to solar

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irradiation. Gene expression for each of the isolates was analyzed independently by comparing

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the gene response of the irradiated sample to that of its corresponding dark control at each time

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point. The results revealed increasing numbers of genes significantly (p < 0.05) upregulated or

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downregulated by ≥ 2-fold in both E. coli PI-7 and DSM1103 as the duration of solar irradiation

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increased (Figure S1). The results also showed an increase in the percentage of total genes that

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were upregulated during the mid- and late decay phase.

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A closer examination showed that E. coli PI-7 upregulated > 50 stress response and

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protection mechanism functions, > 130 cell repair, division and cell wall synthesis functions, and

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>50 virulence functions (virulence factors, motility, membrane transport systems) upon

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prolonged exposure to solar irradiation. E. coli DSM1103 also upregulated genes from the same

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categories. E. coli DSM1103 showed upregulation of ten functions related to stress response, 22


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cell repair functions, and 25 virulence functions. Differential gene expression in each of these

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functional categories is covered in more detail in sections 3.3 to 3.5.

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3.3. Differential gene expression of stress response and protection mechanisms

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Stress response and protection mechanisms (e.g., oxidative stress response, heat shock

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proteins and reactive oxidative species (ROS) scavengers) can be differentially expressed by

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either of the E. coli strains to counteract the effects of solar irradiation.

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In E. coli PI-7, the redox-sensitive transcriptional regulator gene qorR was

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downregulated at the early decay phase (Table 1). Various genes related to oxidative stress

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functions were upregulated at the decay phase, including a catalase, two peroxidases and three

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glutathione-related genes, all known to play a role in ROS scavenging, as well as 39 other genes

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with stress/detoxification-related functions (Table 2 and Table S8A:1-1.5). A superoxide

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dismutase [Cu-Zn] precursor gene in E. coli PI-7 was downregulated from early decay to late

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decay (Table S8A:1.5). In E. coli DSM1103, no response in regulatory oxidative stress genes

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was detected (Table 1). DSM1103 still activated genes in similar oxidative stress response

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categories to E. coli PI-7: a peroxidase, one glutathione-related gene and six other genes with

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stress/detoxification functions. Despite DSM1103 possessing similar numbers of catalase,

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peroxidase

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stress/detoxification-related genes compared with E. coli PI-7, the total number of upregulated

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genes for E. coli DSM1103 in these categories was lower than that observed for PI-7 (Table 2

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and Table S8B:1-1.5).

and

glutathione-related

genes

and

more

than

twice

as

many

other

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Other mechanisms of protection and detoxification were also examined. A regulatory

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gene involved in biofilm formation was upregulated in both E. coli PI-7 and DSM1103 (Table



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1). The marA regulatory gene, encoding a multiple antibiotic resistance protein that is a central

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regulator for efflux pumps, was upregulated in E. coli PI-7 and was present but not upregulated

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in DSM1103 (Table 1). Twenty-two drug and heavy metal efflux pumps and transporters were

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correspondingly upregulated in E. coli PI-7; only one was upregulated in DSM1103 (Table 2).

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Considering this result and the potential of efflux pumps to contribute to antibiotic resistance, the

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effect of efflux pumps in facilitating E. coli PI-7’s prolonged persistence was tested by

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performing simulated solar inactivation trials in the presence of the RND-type efflux pump

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inhibitor, PAβN. This also served as an additional and non-molecular method of confirming the

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transcriptomic findings. The results showed that with PAβN, E. coli PI-7’s lag phase length was

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significantly reduced to less than three hours (p-value = 0.0010), while its half-life was reduced

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to 1.77 ± 0.06 min (p = 0.0360) (Figure 3A). Five-log10 reduction in CFU numbers was achieved

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in under 6.5 h, and 10-log10 of inactivation was achieved by 12 h. E. coli DSM1103 was also

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tested in the presence of PAβN. While the lag phase length did not change significantly, the half-

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life of E. coli DSM1103 significantly decreased to 1.38 ± 0.11 min (p = 0.0446) (Figure 3B).

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This suggests that efflux pump mechanisms have a role against solar irradiation in E. coli

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DSM1103, although it may be less significant role than that in E. coli PI-7.

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3.4. Differential gene expression of cell repair and division mechanisms

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Solar irradiation inactivates microorganisms by inflicting direct and indirect damage on

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various cellular components, compromising DNA and cellular membrane integrity. Therefore,

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gene expression related to cellular repair and division categories was further examined to

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understand the mechanisms employed by both E. coli strains to counteract the inactivating

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effects of solar radiation.


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In E. coli PI-7, the essential DNA repair and maintenance protein gene recA was

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upregulated and the SOS-response repressor gene lexA was downregulated (Table 1). This

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response is required to activate the SOS cellular and DNA repair network. Twenty-eight out of

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66 DNA repair functions, such as three different DNA-damage-inducible protein genes, various

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DNA repair genes, and some exonuclease and endonuclease genes were upregulated in E. coli

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PI-7 (Table 2). Fifteen out of 21 genes with other DNA-related functions, such as a probable

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DNA recombinase and a DNA helicase ruvAB gene (Table S8A:2.2), were also upregulated in

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PI-7 (Table 2). E. coli DSM1103 downregulated its lexA gene but showed no differential

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expression of the recA gene. The observed response for DSM1103 out of 78 DNA repair and 32

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other DNA-related genes was limited to the upregulation of two endonuclease genes (Table

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S8B:2.3), and one gene related to DNA recombination (Table S8B:2.2).

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Functions related to cell wall synthesis, cell division and DNA synthesis were examined.

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In E.coli PI-7, over eighty cell wall synthesis functions were upregulated at different stages of

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exposure to simulated solar irradiation (Table 2). Of those, 28 functions were related to capsular

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and extracellular polysaccharides (Table S8A:5.2). The cell division gene ftsI, which codes for a

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peptidoglycan synthase and belongs to both the cell division and cell wall synthesis categories,

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was upregulated from the mid-lag to the mid-decay phase (Table S8A:3 and S8A:5.1). In

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addition, the replication regulatory protein repA2 gene was downregulated in PI-7 (Table 1), and

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nine DNA replication/synthesis functions (e.g., DNA gyrases, topoisomerases, helicases) were

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also upregulated (Table 2). For E. coli DSM1103, seven cell wall function genes, of which five

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were related to capsular and extracellular polysaccharides, were upregulated from the mid- to

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late decay phase (Table 2). In the cell division and replication category, one protein-coding gene

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related to septum formation and another putative replication protein were upregulated (Table 2).



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Cells can also adopt dormancy or form persister cells in the event that their repair

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mechanisms are not sufficient to counteract oxidative stress. E. coli PI-7 upregulated 19 out of

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57 genes with functions related to dormancy and persister cell formation (Table 2). One possible

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way to achieve dormancy is through programmed cell death and toxin-antitoxin systems. The

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genes for the YoeB-YefM pair, the only complementary toxin-antitoxin pair detected for E. coli

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PI-7 in this experiment, were upregulated at late decay (Table S8A:6.1). Furthermore, the

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dormancy and persister-cell-related gene hipB in E. coli PI-7 was upregulated at the mid- and

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late decay phase (Table S8A:4), as were other transcription factors, heat/cold shock genes, and

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SOS-related genes implicated in persister formation. For E. coli DSM1103, seven out of 44

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dormancy and persister cell formation genes were upregulated (Table 2, and Table S8B:4). Five

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genes with toxin-antitoxin functions (e.g., antitoxin YefM, three genes with toxin Ldr functions)

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were upregulated at the mid- and late decay phase (Table S8B:6.1), but the available annotation

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did not reveal whether there were any complementary toxin-antitoxin pairs.

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3.5. Differential gene expression of virulence functions

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Virulence-related functions such as virulence signaling, adhesion and invasion functions

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were examined for their gene expression response to simulated solar irradiation, particularly for

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the purpose of understanding the risk associated with E. coli PI-7’s virulence and prolonged

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persistence. E. coli PI-7 showed upregulation of the regulatory gene rpoN, which encodes a

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sigma factor known to play a role in bacterial virulence (Table 1). It also upregulated 25 genes

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with virulence functions including four with cell signaling functions related to virulence at the

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mid-decay phase (Table S8A:6.3-7.3) and a virulence protein gene that reached 20-fold

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upregulation at high RPKM levels (Table S8A:7, and Table S9A:7). E. coli PI-7 also upregulated


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genes with two flagellar regulatory functions and 29 functions related to flagella and motility

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(Table 2). For E. coli DSM1103, there was a detectable virulence response in the upregulation of

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six virulence-related functions (Table 2, and Table S8B:6.3-7.3).

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An earlier study to characterize the genomic and plasmidic content of E. coli PI-7

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revealed a repertoire of pathogenic traits including colonization factor antigen I fimbriae

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(CFA/I), accessory colonization factor acfD precursor, the type III secretion system and various

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putative virulence factors13. These virulence-associated traits are unique to E. coli PI-7 and are

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not present in E. coli DSM1103’s genome. Both CFA/I fimbrial subunit genes were upregulated

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upon solar irradiation (Table 1), with one upregulated at mid-decay and the other throughout the

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decay phase, and 25 other genes with fimbriae-related functions were upregulated as well (Table

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2). Accessory colonization factor acfD precursor gene was upregulated at late decay alongside

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other adhesion functions. For example, the fimH adhesin gene was upregulated at late decay, and

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four other genes with adhesion functions were upregulated at various decay phases (Table

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S8A:7.1). The four genes that belong to the type III secretion systems were also upregulated, two

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at the mid-decay and the other two at the late decay phase (Table 2, and Table S8A:9.5).

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Moreover, upregulation of functions related to the conjugative transfer of E. coli PI-7’s IncF

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blaNDM-1-carrying plasmid was also observed (Table S8A:9.3), along with over 30 functions

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belonging to secretion systems II, VI, VII and VIII (Table S8A:9.4-9.8). E. coli DSM1103 has

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no functions belonging to the type III secretion system, but genes related to the type VI and VII

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secretion systems were upregulated at the late decay phase (Table S8B:9.6-9.7).

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3.6. RNA-seq confirmation using reverse transcription quantitative PCR (RT-qPCR)



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To validate the RNA-seq results, RT-qPCR was performed on RNA from the early and

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mid-decay phase for each of the two E. coli strains. For eight out of ten data points obtained for

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E. coli PI-7, the fold changes in gene expression from RNA-seq and RT-qPCR were in good

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agreement (Figure S2A-E). There were slight exceptions in two instances. The RNA-seq results

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showed significant upregulation of the gene hflC at both early and mid-decay, but only slight

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upregulation for the same gene was observed in mid-decay when examined using RT-qPCR

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(Figure S2B). For the second exception, RT-qPCR showed downregulation of the gene marA at

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mid-decay, while RNA-seq showed upregulation (Figure S2D). However, the upregulation in the

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RNA-seq result was not statistically significant. For E. coli DSM1103, four out of eight data

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points were in good agreement between RT-qPCR and RNA-seq results (Figure S3B-D). For

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those data points that showed discrepancies, RT-qPCR consistently showed downregulation of

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the tested genes while RNA-seq showed upregulation of these genes. However, it is to be noted

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that most of the upregulation obtained by RNA-seq did not have significant fold-change values

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except for the gene ftsI at mid-decay (Figure S3A).

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4. Discussion

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Treated municipal wastewater has been increasingly recognized as a potential source for

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the dissemination of emerging contaminants including antibiotic-resistant bacteria (ARB) into

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the environment, even after WWTP processes 3, 4, 32-35. The ability of ARB to survive and persist

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in treated effluents can be a concern if the treated wastewaters are to be reused for agricultural

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irrigation. To mitigate concerns arising from ARB in treated wastewater, sunlight irradiation can

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be utilized as one of the final barriers to prevent dissemination of contaminants into the

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environment during agricultural irrigation events. This is because the full spectrum of sunlight


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includes radiation in the UV-A and UV-B spectra, which are known to impose biocidal effects

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on microorganisms through direct and indirect mechanisms

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radiation penetrates the cell walls of microorganisms and is absorbed by nucleic acids, resulting

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in dimerization of adjacent cytosines and thymines 39. In the indirect mechanisms, endogenous or

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exogenous photosensitizers absorb UV light

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(ROS) that can damage the cellular membrane, nucleic acids, proteins or other cellular materials

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42, 43

40-42

36-38

. In the direct mechanisms, UV

and generate strong reactive oxidative species

.

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In this study, exposure to simulated solar irradiance achieved ≥ 5-log10 reduction in the

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numbers of viable E. coli cells, but the response curves and irradiance doses required for the

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reduction differed between the two isolates. Specifically, E. coli PI-7 exhibited an extended lag

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phase before the onset of detectable inactivation compared to E. coli DSM1103, and it also had a

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longer decay half-life and a tailing zone at late-decay with significantly slower inactivation in

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35% of PI-7 inactivation trials. There are limited studies that demonstrate the differences in the

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inactivation kinetics of different strains of E. coli upon solar irradiation. However, our

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observations are in agreement with the few studies available that compared the differences in the

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inactivation kinetics of strains within other bacterial species, where it was found that different

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strains of Acinetobacter johnsonii and Halomonas salina exhibited variations in their survival

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rates after UV-B irradiation 44, 45. Variations in the decay kinetics among different strains of the

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same bacterial species may be attributed by factors such as genome size, nucleotide base

412

composition and genomic content

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genomic contents and, therefore, may have differences in their molecular responses to the

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simulated solar irradiance. To investigate the underlying mechanism of each isolate’s response to

46

. In this study, the two E. coli isolates tested have different



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simulated solar irradiance, the gene expression of the irradiated samples was compared with that

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of the dark controls at the various points of the inactivation curve for each E. coli strain.

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The transcriptomic results showed that a large portion of the genes that were

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differentially regulated by E. coli PI-7 were shared between genomes of the two E. coli strains.

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Furthermore, despite E. coli DSM1103 possessing the larger genome size and higher number of

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unique gene sequences and metabolic functions, E. coli PI-7 differentially regulated a larger

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number of its available annotated genes compared with DSM1103, and it also exhibited a higher

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percentage of gene upregulation. A closer examination of specific gene categories revealed that

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E. coli PI-7 upregulated a large number of genes involved in DNA recombination and repair,

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suggesting that E. coli PI-7’s ability to activate a wide arsenal of DNA modification and DNA-

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damage repair functions is a key factor contributing to the prolonged persistence of this isolate

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under solar irradiance. DNA recombination and repair mechanisms are inducible as part of the

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SOS regulon

428

downregulated in both E. coli strains. In addition, a large number of genes with cell wall

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functions, specifically ones related to the cell capsule and extracellular polysaccharides, were

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upregulated in E. coli PI-7. The cell membrane is considered one of the key targets of damage

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induced by solar irradiance, as it is the first point of contact with exogenous photosensitizers.

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Active synthesis of cell wall components is indicative of continuous damage repair in E. coli PI-

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7. Extracellular polysaccharides may also encapsulate the cells and provide a protective layer to

434

shield against solar irradiance

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biofilm formation-related genes, and the biofilm matrix can also serve as a physical shield

436

against solar irradiance.

47

, and the associated SOS-response repressor gene lexA was observed to be

48, 49

. Similarly, both E. coli strains upregulated a number of


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In addition to active cell repair, the longer persistence of E. coli PI-7 compared to E. coli

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DSM1103 can be accounted for by the upregulation of oxidative stress response and protective

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mechanisms. Interestingly, superoxide dismutase, which is known to convert superoxide O2- into

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less damaging oxygen and hydrogen peroxide

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suggesting that the superoxide O2- ROS was not a key mechanism of damage in this study.

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Instead, genes implicated in the response to hydrogen peroxide radicals were upregulated in E.

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coli PI-7. Catalase is known to be a hydrogen peroxide scavenger that converts hydrogen

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peroxide to water and oxygen

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decay phases in E. coli PI-7, but another was downregulated at mid-lag and early decay.

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Although catalase scavenges hydrogen peroxide and protects bacterial cells against UV damage,

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excess catalase can act as a photosensitizer and cause increased sensitivity to UV damage when

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intracellular antioxidant moieties are limited

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production may have been undertaken by E. coli PI-7, explaining the mixed response and

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upregulation of different catalase genes at varying decay phases. Cytochrome c peroxidase,

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another enzyme known to reduce hydrogen peroxide to water 56, was also upregulated in E. coli

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PI-7. Although no measurements were performed to determine the ROS abundance, it can be

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inferred from these findings that hydrogen peroxide may be the predominant ROS causing solar

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inactivation in this study. No evidence for a hydrogen peroxide-specific response was observed

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in E. coli DSM1103, and this may have contributed to the faster inactivation displayed by E. coli

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DSM1103. Our results are in good agreement with a microarray study on the response of

457

Enterococcus faecalis to natural sunlight. Despite being a gram-positive bacterium, E. faecalis

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also demonstrated upregulation in genes related to DNA repair and replication, peptidoglycan

459

and cell wall, cell redox homeostasis and oxidative stress functions (including thioredoxin- and

51-53

50

, was downregulated in both E. coli strains,

. One catalase/peroxidase gene was upregulated during the

42, 54, 55

. Hence, tight regulation of catalase



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glutathione-related ones) upon solar irradiation. However, upregulation of superoxide dismutase

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was observed in E. faecalis, suggesting that superoxide O2- ROS may be implicated as a key

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mechanism of cell damage against this bacterium 57. This is in contrast to our findings obtained

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from both E. coli strains.

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In addition to oxidative stress response and ROS scavenging, E. coli PI-7 activated a

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large number of multidrug efflux pumps and transporters, suggesting that cellular detoxification

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might also play a role in the response to solar irradiance. Simulated solar inactivation trials on E.

467

coli PI-7 in the presence of efflux pump inhibitor PAβN showed a great decrease in PI-7’s

468

persistence as it significantly shortened its lag phase and half-life and achieved higher overall

469

log10 reduction in CFU numbers over a shorter duration of time. E. coli DSM1103 upregulated a

470

single efflux pump function, and, while the effect of PAβN was less extreme on E. coli

471

DSM1103, it also showed a decreased half-life. Although efflux pumps’ effects on inactivation

472

have not been reported in solar irradiation studies, similar findings have been reported among

473

microbial communities that were exposed to chlorination. Similar to the indirect inactivation

474

mechanism of solar irradiation, chlorination results in oxidative stress. A study assessing the

475

effect of chlorination found induced transcription of genes encoding major virulence factors in S.

476

aureus 58 and chlorine-associated induction of antibiotic resistance in eight antibiotic-resistant A.

477

baumannii pathogen isolates 59. Tetracycline resistance conferred by an efflux pump mechanism

478

encoded by the gene tetZ was also documented to be enriched in bacterial DNA recovered from

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chlorinated WWTP effluent samples 4.

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In addition to active repair mechanisms and stress response and detoxification, dormancy 60

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and formation of persister cells are documented responses to stress

482

useful defense mechanisms in the event that active cell repair remains insufficient to mitigate the

. These are particularly


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UV-induced damage. Evidence suggesting formation of dormant or persister cells was observed

484

during the late phase of decay, in which E. coli PI-7 exhibited an upregulation in toxin-antitoxin

485

systems for programmed cell death and a number of dormancy and persister-cell related genes.

486

These results may account for the tailing effect seen at the end of E. coli PI-7’s inactivation

487

curve, suggesting that a subpopulation of the E. coli cells entered into dormancy and persisted

488

despite continuing irradiation. The persistence of E. coli PI-7 subpopulations may be a concern

489

since E. coli PI-7 showed upregulation of virulence functions, a suite of genes involved in the

490

replication and conjugative transfer of its NDM-1-carrying IncF plasmid and the CTX-M-15-

491

carrying IncA plasmid.

492

The large number of virulence genes that were upregulated in E. coli PI-7 draws parallels

493

with reported observations of pathogens activating virulence genes to evade the host’s immune

494

response during infection61. When pathogenic strains invade a host, the host’s immune response

495

triggers production of ROS to induce cell apoptosis, which approximates the situation

496

experienced by bacterial cells during solar irradiation61. It is therefore likely that the upregulation

497

of virulent traits in E. coli PI-7 (a pathogenic strain) is a consequence of solar irradiation,

498

undertaken in response to the external stress conditions. An alternative possible interpretation

499

would be that the virulence traits contribute to defense against solar irradiation and/or are

500

oxidative stress response mechanisms, hence resulting in extended decay kinetics for E. coli PI-

501

7. However, this hypothesized role for virulence genes in slowing decay kinetics remains to be

502

further elucidated.

503

Inactivation of the two E. coli strains in wastewater was tested to examine the response in

504

more realistic matrices. It was found that wastewater, compared with buffer solution, caused a

505

significant increase in the half-life of both E. coli strains. Specifically, the half-life of E. coli PI-7



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was significantly longer in chlorinated WWTP effluent, while the half-life for E. coli DSM1103

507

was significantly longer in non-chlorinated effluent. The slower decay experienced by the E. coli

508

strains may be due to the high alkalinity present in the wastewater matrix (Table S5).

509

Bicarbonates (HCO3−) present in water can react with hydroxyl radicals producing CO3•−, which

510

has a slower reaction with organic molecules when compared to •O 19, 62. On the other hand, the

511

high SUVA content in both types of wastewater indicates the presence of dissolved organic

512

matter, which may have shielded the microbial cells from sunlight irradiation or may have

513

cleaved into smaller, biologically labile organic compounds that can in turn stimulate further

514

bacterial growth

515

observed even though the examined wastewater samples were filtered to remove turbidity. In

516

actual instances of wastewater with high turbidity, the scattering effect of particulates would

517

likely result in an even longer half-life than that observed in this study. Furthermore, in the case

518

of E. coli PI-7, its longer half-life in the chlorinated effluent could be in part related to its

519

upregulation of efflux pump and antibiotic resistance functions, which may have helped maintain

520

homeostasis, not only against chlorine but also against ROS generated during irradiation.

521

Induction of these genes has also been reported in other bacterial species upon exposure to

522

chlorine

523

efflux pump mechanisms was determined in the total microbial community recovered from

524

chlorinated effluent compared to pre-chlorinated effluent 4. These observations suggest that these

525

traits may act as defense mechanisms against chlorine and other deleterious elements, hence

526

resulting in the longer persistence of E. coli PI-7 in the chlorinated effluent.

58, 59

63, 64

. The longer half-life of both E. coli strains in a wastewater matrix was

, while an increase in the abundance of antibiotic resistance genes associated with

527

In summary, this study revealed differences in the solar inactivation kinetics of the

528

virulent antibiotic-resistant E. coli PI-7 strain and a non-virulent and the less antibiotic-resistant


Page 25 of 38


529

commensal E. coli DSM1103. The results showed that a minimum of half a day’s exposure to

530

solar irradiation is required to overcome the lag phase of E. coli in the buffer solution and treated

531

wastewater prior to achieving inactivation. Although solar irradiation remains useful and

532

effective in reducing the number of E. coli cells by more than 5-log10, transcriptomics revealed

533

differences in the overall upregulation of regulatory, protective and repair mechanisms between

534

both E. coli strains, leading to different efficacies in damage sensing and response.

535

Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation

536

during the late decay phase, which may have accounted for the prolonged persistence of E. coli

537

PI-7. More importantly, this study showed that both E. coli strains displayed upregulation of

538

virulence functions, with a wider arsenal of such genes being upregulated in the virulent E. coli

539

PI-7, upon solar irradiation. The current guidance on the quality of treated wastewater for use in

540

agricultural irrigation remains limited to assessing the abundance of fecal coliforms. Our

541

findings suggest a need to expand efforts to monitor the presence and abundance of ARB,

542

particularly for those bacterial pathogens that are commonly associated with foodborne diarrheal

543

diseases, in the treated wastewater that is to be reused.

544 545

Acknowledgements

546

This study is supported by KAUST baseline funding BAS/1/1033-01-01 awarded to P.-Y. Hong.

547

The authors would to thank Dr. Shengkun Dong and Professor Helen Nguyen for providing

548

training to N.A.-J. on the use of the solar simulator in the earlier phase of this project; Dr, Inhyuk

549

Kwon and Professor Roderick Mackie for providing training to N.A.-J. on transcriptomics

550

analysis; and Dr. Muhammad Raihan Jumat for providing assistance with reverse transcription.

551

The authors would also like to express gratitude to Mr. George Princeton Dunsford for granting



Page 26 of 38

552

access to the wastewater samples and to Dr. Moataz El Ghany for ideas pertaining to E. coli

553

transcriptomics.

554 555

Supplementary Information Available

556

Methods: water quality testing, solar inactivation experiments, statistical comparisons of

557

inactivation curves, RNA extraction and sequencing, RNA-seq analysis, RNA-seq mapping

558

quality, cDNA synthesis, RT-qPCR confirmation experiments. Tables: genomic comparison

559

overview, RNA-seq mapping quality, list of primers used for RT-qPCR, wastewater quality,

560

inactivation kinetics in wastewater, genomic comparison in specific functional categories, fold

561

change of differentially regulated genes in select categories, RPKM values of differentially

562

regulated genes in select categories. Figures: percentages and numbers of differentially regulated

563

genes, PI-7 and DSM1103 inactivation with the efflux pump inhibitor, RNA-seq and RT-qPCR

564

fold change for PI-7 and DSM1103.


Page 27 of 38

565 566 567

568 569 570 571


Table 1. Regulatory genes with differential gene expression responses in stress response, cell repair and virulence categories PI-7 DSM 1103 a) Stress response NA QorR, redox-sensing transcriptional regulator + + Biofilm formation regulator + o MarA, multiple antibiotic resistance protein b) Cellular repair + o RecA LexA Repressor NA Replication regulatory protein repA2 c) Virulence + NA Predicted regulator of CFA/I fimbriae + NA FimB type 1 fimbriae regulatory proteins + NA FimE type 1 fimbriae regulatory proteins + o FliA, RNA polymerase σ factor for flagellar operon + o FlhC, flagellar transcriptional activator o FlgM, negative regulator of flagellin synthesis + o RpoN, σ54 RNA polymerase (+): upregulated; (-): downregulated; (o): present in genome but with no significant fold change in expression; (NA) not found in the genome. This list of regulatory genes is selected from the full list of genes with significant fold change in the selected categories (See Table S8).



572 573

Page 28 of 38

Table 2. Numbers of upregulated genes out of the total number of present genes in stress response, cell repair and virulence categories

574

575 576 577 578 579 580 581 582 583 584 585

PI-7 DSM 1103 a) Stress response Biofilm 6/13 1/4 Catalases 1/2 0/2 Peroxidases 2/3 1/2 Glutathione 3/22 1/20 Other stress/detox 39/70 6/171 Multidrug and heavy metal efflux pumps/transporters 22/43 1/19 b) Cellular repair DNA repair 28/66 2/78 Cell division 17/38 1/36 Cell wall synthesis 87/191 7/192 DNA synthesis 9/14 1/15 Other DNA 15/21 4/32 Persisters 19/57 7/44 c) Virulence Virulence 25/41 6/38 Fimbriae/pili 30/53 13/42 Flagella and motility 29/61 6/60 Type III secretion system 4/4 0/NA This list of regulatory genes is selected from the full list of genes with significant fold change in the selected categories (See Table S8). Genes were counted for inclusion in this table if they were upregulated at any of the examined time points; total numbers are the total numbers of relevant genes that were found in the entire genomes of the E. coli isolates.


Page 29 of 38

586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604


Figure legends Figure 1. Inactivation curves of E. coli PI-7 and DSM1103 under simulated solar irradiation in buffer solution. E. coli PI-7 exhibits a longer lag phase than DSM1103 prior to decay. The irradiance rate at 280-700 nm was 27.86 J/cm2/h. Figure 2. Inactivation curves of E. coli PI-7 and DSM1103 under simulated solar irradiation in effluent and chlorinated effluent. Irradiated E. coli PI-7 and DSM1103 in chlorinated effluent and effluent, respectively, showed a longer half-life than the same strains in buffer. Figure 3. Inactivation curves of (A) E. coli PI-7 and (B) DSM1103 in phosphate-buffered saline and in the absence or presence of 50 µg/mL efflux pump inhibitor (PAβN). EPI: efflux pump inhibitor. One-way ANOVA on log reduction in dark samples ± EPI showed that 50 µg/mL PAβN had no toxic effect on the bacteria (p-value = 0.92). E. coli PI-7 + EPI showed reduced lag phase length and half-life vs. PI-7 - EPI. DSM1103 with EPI showed no significant change in lag phase length but a reduced half-life compared with DSM1103 without EPI.



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(A) PI-7 2

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4

5

6

7

1.00

0.00

0.00

-1.00

-1.00

-2.00

-2.00

-3.00

-3.00

-4.00

-4.00 R² = 0.9115

-5.00

-5.00

-6.00

-6.00

-7.00

-7.00

-8.00

-8.00

-9.00

-9.00

0

50

Fluence (J/cm2)

Dark control

Page 38 of 38

Dark control

+EPI Irradiated

100 Fluence (J/cm2)

Irradiated

150

+EPI Dark control

+EPI Irradiated

Lag phase length (h)

Lag phase fluence (J/cm2)

Decay constant

Half-life length (min)

Half-life fluence (J/cm2)

6.64 ± 0.63

184.96 ± 17.55

-14.96 ± 2.61

2.85 ± 0.46

1.32 ± 0.21

Inactivation and Gene Expression of a Virulent Wastewater

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