Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole.

Escherichia coli is the most frequent agent of urinary tract infections in humans. The emergence of uropathogenic multidrug-resistant (MDR) E. coli strains that produce extended spectrum β-lactamases (ESBL) has created additional problems in providing adequate treatment of urinary tract infections. We have previously reported the antimicrobial activity of 1,8-cineole, one of the main components of Rosmarinus officinalis volatile oil, against Gram negative bacteria during planktonic growth. Here, we evaluated the antibiofilm activity of 1,8-cineole against pre-formed mature biofilms of MDR ESBL-producing uropathogenic E. coli clinical strains by carrying out different technical approaches such as counting of viable cells, determination of biofilm biomass by crystal violet staining, and live/dead stain for confocal microscopy and flow cytometric analyses. The plant compound showed a concentration- and time-dependent antibiofilm activity over pre-formed biofilms. After a 1 h treatment with 1% (v/v) 1,8-cineole, a significant decrease in viable biofilm cell numbers (3-log reduction) was observed. Biofilms of antibiotic-sensitive and MDR ESBL-producing E. coli isolates were sensitive to 1,8-cineole exposure. The phytochemical treatment diminished the biofilm biomass by 48-65% for all four E. coli strain tested. Noteworthy, a significant cell death in the remaining biofilm was confirmed by confocal laser scanning microscopy after live/dead staining. In addition, the majority of the biofilm-detached cells after 1,8-cineole treatment were dead, as shown by flow cytometric assessment of live/dead-stained bacteria. Moreover, phytochemical-treated biofilms did not fully recover growth after 24 h in fresh medium. Altogether, our results support the efficacy of 1,8-cineole as a potential antimicrobial agent for the treatment of E. coli biofilm-associated infections.

Introduction

Uropathogenic Escherichia coli is the most common cause of urinary tract infections, accounting for approximately 80% of infections [1]. The routine therapy of urinary tract infections is based on the use of antibiotics such as β-lactams, trimethoprim, nitrofurantoin and quinolones in many countries. Over-use and misuse of these antibiotics increase the development of resistant bacteria [2]. Particularly, the emergence of uropathogenic multidrug-resistant (MDR) E. coli strains that produce extended spectrum β-lactamases (ESBL) is a serious global health problem, since it can cause prolonged hospital stay, increasing morbidity, mortality, and health care costs [3]. ESBLs are a group of β-lactamase enzymes that confer resistance to third generation cephalosporins, such as ceftazidime and ceftriaxone. Resistance genes coding for β-lactamases are often located on plasmids which also harbor resistance genes for non- β-lactam antibiotics such as aminoglycosides and trimethoprim-sulfamethoxazole [4]. Therefore, ESBL producing bacteria are commonly MDR, leaving limited antibacterial options.

Uropathogenic E. coli forms multicellular communities known as biofilms, residing in the bladder epithelium and also on urinary catheters [5]. Bacterial biofilms are microbial communities of cells attached to a biotic or abiotic surface and embedded in a self-produced extracellular polymeric matrix [6]. Bacteria grown in biofilms are significantly more resistant to antibiotics than planktonic cells [7]. Varied mechanisms have been proposed to elucidate the high antibiotic resistance of biofilms including restricted antibiotic penetration, decreased growth rates and metabolism, and induction of cell biofilm–specific phenotypes known as persister cells [8]. The increased resistance of uropathogenic E. coli to antibiotics along with the bacterial ability to form biofilms cause recurrence and chronicity of urinary tract infections [5].

In the era of increasing antibiotic resistance, the search of new antimicrobial agents effective against pathogenic bacteria in their two ways of life, planktonic and biofilm stage, is a priority need in the clinical practice [9]. Volatile oils derived from aromatic and medicinal plants, such as rosemary (Rosmarinus officinalis), peppermint (Mentha piperita), thyme (Thymus vulgaris), fennel (Foeniculum vulgare), are reported to be effective against Gram-negative and Gram-positive bacteria, viruses, and fungi [10]. These plant volatile oils are complex organic metabolites with lipophilic characteristics. The specific role of individual compounds as responsible for the antimicrobial effect has not been extensively studied [11]. In a previous study, we reported one of the main constituents of rosemary volatile oil, the monoterpene 1,8-cineole (also known as eucalyptol), which exhibited a marked antibacterial activity against E. coli ATCC35218 strain [12]. At the minimum inhibitory concentration (MIC) [0.8% (v/v)], 1,8-cineole showed bactericidal effect on planktonic E. coli cells, with membrane disruption as the bactericidal mechanism identified. Other authors reported MICs of 1,8-cineole for E. coli strains ≥ 0.8% (v/v) [13-15]. Nevertheless, the effect of this phytochemical on E. coli biofilms has not been extensively explored. In particular, little is known about the effect of 1,8-cineole on bacterial viability of MDR ESBL-producing uropathogenic E. coli growing in biofilms.

Thus, the main goal of this study was to analyze the antibiofilm activity of 1,8-cineole against mature biofilms of MDR ESBL-producing uropathogenic E. coli clinical strains by evaluating its effect on biofilm biomass and cell viability.

Materials and methods

Bacterial strains and inoculum preparation

Escherichia coli strains used in this study were isolated from adult patients and are described in Table 1. E. coli strains named Ec AM were isolated from urinary samples collected from patients admitted to a medical center at Buenos Aires (Argentina) between 2017 and 2018 [16]. Strain Ec07 was isolated from a patient with polymicrobial CAUTI at Hospital Pirovano (Buenos Aires City, Argentina) [17]. Microbiological identification and antimicrobial susceptibility testing were carried out by standard methods. In vitro susceptibility tests were interpreted based on CLSI breakpoints [18]. E. coli strains were examined for ESBL production by a double-disk synergy test using ceftazidime, cefotaxime and cefepime with and without clavulanic acid according to CLSI guidelines [18]. E. coli clinical strains used in this study were isolated as part of routine clinical hospital procedures to diagnose infection and hence ethical approval was not required, according to the corresponding institutional guidelines.

Table 1

E. coli strains used in this study.

Strain	Description	Antibiotic resistancea	Source
Ec ATCC25922	Urinary isolate	None	ATCC
Ec AM3	Urinary isolate (ESBL producer)	NIT, TMS, CIP, AMC, CTX, CAZ, CEF	This work
Ec AM4	Urinary isolate (ESBL producer)	NIT, TMS, CIP, AMC, CTX, CAZ, CEF	This work
Ec AM5	Urinary isolate (ESBL producer)	CIP, AMC, CTX, CAZ, CEF	This work
Ec AM6	Urinary isolate (ESBL producer)	TMS, CIP, AMC, CTX, CAZ, CEF, GEN, AKN	This work
Ec AM7	Urinary isolate (ESBL producer)	TMS, CIP, AMC, CTX, CAZ, CEF, GEN, AKN	This work
Ec AM8	Urinary isolate (ESBL producer)	NIT, CIP, AMC, CTX, CAZ, CEF	This work
Ec AM9	Urinary isolate (ESBL producer)	TMS, CIP, CTX, CAZ, CEF	This work
Ec AM10	Urinary isolate (ESBL producer)	CIP, AMC, CTX, CAZ, CEF	This work
Ec AM12	Urinary isolate (ESBL producer)	NIT, TMS, CIP, AMC, CTX, CAZ, CEF, GEN	This work
Ec 07	Urinary isolate (ESBL producer)	AMP, CIP, CTX, CAZ, CEF, CEP, GEN, NAL,	[17]

a AMP, ampicillin; AMC, amoxicillin- clavulanic acid; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CEF, cephalothin; NIT, nitrofurantoin; TMS, trimethoprim/ sulfamethoxazole; AKN, amikacin; GEN, gentamicin; NAL, nalidixic acid; CIP, ciprofloxacin.

ATCC, American Type Culture Collection; ESBL, extended spectrum β-lactamase.

Isolates were maintained in the laboratory as frozen stocks (at –80°C) in Luria-Bertani (LB) broth supplemented with 15% glycerol. Inocula for assays were prepared as follows. Strains were streaked on Tryptic Soy Broth (TSB)-agar plates and grown overnight at 37°C. Subsequently, individual colonies were used to inoculate TSB (3 ml) and were incubated overnight at 37°C and 200 rpm. Then, each inoculum was properly diluted in M9 minimal medium supplemented with 0.8% glucose in order to obtain 107 cells ml-1.

Biofilm formation assays

Bacterial inocula in M9 supplemented with 0.8% glucose (1 × 107 cells ml-1) were placed in 96-well (200 μl per well) or 24-well (1 ml per well) polystyrene plates (DeltaLab, Barcelona, Spain) and incubated statically at 37°C. Adhesion to polystyrene surface was allowed for 3 h and then the medium was replaced every 24 h for up to 3 d. At selected time points, biofilms developed in 96-well plates were washed three-times with sterile 0.9% NaCl before biomass quantification by crystal violet staining (absorbance measurement at 595 nm) [19]. All crystal violet assays were performed in technical quadruplicate and, for each plate, four wells were used as blanks containing sterile growth medium. Experiments were done in biological triplicates Biofilm biomass levels were classified as highly positive (A595 ≥ 1), low-grade positive (0.2 ≤ A595 ≤ 1), or negative (A595 ≤ 0.2) [20].

For quantification of cultivable cells, biofilms developed in 24-well plates were washed with sterile 0.9% NaCl before mechanical disruption from the surface as previously described [21]. The bacterial suspensions obtained were serially 10-fold diluted, plated on TSB-agar plates, and grown for 16 h at 37°C for enumeration of colony forming units (cfu). Experiments were done in biological triplicates and technical duplicates were performed.

Determination of 1,8-cineole minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of 1,8-cineole was determined by using the broth microdilution method, with minor modifications [12,18]. In brief, assays were performed in 96-well plates and using M9 supplemented with 0.8% glucose and containing 0.5% Tween 80 (200 μl final volume). Tween 80 (0.5%) was added to the medium to enhance phytochemical solubility [22,23]. 1,8-cineole (Sigma, MO, USA) dilutions (0.25–2%, v/v) in medium were prepared from an 80% (v/v) pure compound solution in ethanol and mixed with each bacterial strain at an initial inoculum of 1 × 106 cells ml-1. The plates were then incubated at 37°C for 24 h and bacterial growth assessed by measuring the absorbance at 595 nm. The MIC was defined as the 1,8-cineole concentration able to inhibit 90% of bacterial growth after 24 h incubation. As previously shown [12,22,23], bacterial growth was not affected by addition of 0.5% Tween 80 (A595nm 0.824 ± 0.046 vs. 0.839 ± 0.031 in the absence and in the presence of Tween 80, respectively). Controls containing 0.5% (v/v) ethanol, that correspond to the amount of ethanol present in the highest concentration of phytochemical tested [1,8-cineol 2% (v/v)] did not significantly inhibit bacterial growth (less than 2% inhibition). Experiments were done in biological triplicates and technical triplicates were performed.

Biofilm susceptibility to 1,8-cineole

Mature biofilms (3 d-old) were washed with 0.9% NaCl, then, the indicated concentration of 1,8-cineole in M9 supplemented with 0.8% glucose and 0.5% Tween 80 were carefully added on top of the biofilms, and the plates were incubated statically at 37°C. Controls (untreated) were carried out by replacing the culture medium by fresh medium. Medium supplemented with 0.5% Tween 80 was also assayed as control, giving similar result than medium without this surfactant. Vehicle controls were assessed using the ethanol concentrations corresponding to each phytochemical dilution used in medium supplemented with 0.5% Tween 80 (ethanol concentrations of 0.03, 0.06, 0.12, 0.25, 0.50%, v/v, corresponding to 1,8-cineole concentrations of 0.12, 0.25, 0.50, 1.00, 2.00%, v/v, respectively). After 15 to 180 minutes of incubation, the medium was removed, biofilms washed with 0.9% NaCl and biofilm biomass and cell viability was determined as explained before.

Assays to investigate biofilm regrowth after phytochemical treatment were performed as follow. Mature biofilms (3-d-old) developed in 24-well plates were treated with 1% 1,8-cineole (v/v) for 1 h. Vehicle controls were assessed using 0.25% ethanol. After treatment, biofilms were washed three-times with 0.9% NaCl and then fresh M9 medium supplemented with 0.8% glucose was added to the wells and plates were incubated at 37°C. At 6 h and 24 h, the medium was removed, biofilms were washed with 0.9% NaCl, and cell viability was determined as explained before. Experiments were done in biological triplicates and technical duplicates were performed.

Biofilm imaging

Ec AM7 biofilms were formed on 18-mm glass coverslips, as described above. Three-days-old biofilms were treated with 1% (v/v) 1,8-cineole during 1 h. Controls were carried out as explained above. Biofilms were further stained using the live/dead BacLightTM Bacterial Viability Kit (Thermo Fisher Scientific, Waltham, MA, USA) containing SYTO®9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which was handled following the provider’s recommendations. Observation of biofilms was done using a Carl Zeiss LSM 800 confocal laser scanning microscope (Zeiss, Oberkochen, Germany). For each biofilm, three image stacks were taken with a z-step size of 1 μm. Unstained and single-stained slices for each dye were used to monitor and subtract all respective background signals. The Zeiss ZEN Microscope Software version 3.0 was used for generation of orthogonal and 3D images. COMSTAT 2.1 (www.comstat.dk) [24,25] and the ImageJ software distribution FIJI [26] were utilized for biomass calculations and to quantify the viable (SYTO®9; green), dead (propidium iodide; red) and colocalized (SYTO®9 + propidium iodide; yellow) parts of the biofilms from the image z-stacks. Colocalized fluorescence was defined as part of propidium iodide staining, as the dye was able to penetrate the membrane. As it did not completely remove SYTO®9, it was subtracted from SYTO®9 staining.

Evaluation of biofilm-detached cells

Ec AM7 biofilms grown for 3 d were treated with 1% (v/v) 1,8-cineole during 1 h. Controls were carried out as explained above. Bacteria in the surrounding media (~ 1 ml) were taken and centrifuged, and the pellet was resuspended in 1 ml of 0.9% NaCl. For quantification of viable cells, bacterial suspensions were serially 10-fold diluted, plated on TSB-agar plates, and grown for 16 h at 37°C for cfu enumeration. Additionally, cell viability was assessed by flow cytometry (BD FACSCanto II, Becton, Dickinson and Co., NJ, USA), using the live/dead BacLightTM Bacterial Viability Kit (Thermo Fisher Scientific, Waltham, MA, USA), as described [27]. As a control, bacterial cells were killed by incubating for 60 min at 28°C in 70% isopropanol. Flow cytometry analysis of propidium iodide and SYTO®9 co-stained bacteria was carried out using FlowJo software v10.0.7.

Statistical analysis

Statistical significance between control and 1,8-cineole-treated samples was determined with either paired Student´s t-test (one-tailed) or the one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test using GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA). Differences were considered significant when P values were less than 0.05.

Results

Biofilm formation ability of multidrug-resistant ESBL-producing uropathogenic E. coli clinical strains

Urinary tract infections caused by multidrug-resistant (MDR) E. coli strains that produce extended spectrum β-lactamases (ESBL) have become an increasing health problem. An additional virulence factor reported for uropathogenic E. coli strains is biofilm formation [1]. Initially, biofilm formation ability of ten MDR ESBL-producing E. coli clinical isolates from urine patients was assessed over time (1–3 d) by determining biofilm biomass with crystal violet staining. Three isolates showed a biofilm biomass that increased over time (Fig 1), whereas seven strains were negative for biofilm production (A595nm ≤ 0.198 at d 3). At d 3, Ec AM7 was identified as highly-positive biofilm producer (A595nm of 2.684 ± 0.553), whereas EcAM10 and Ec07 were low-grade biofilm producers (A595nm of 0.448 ± 0.144 and 0.701 ± 0.109, respectively). Also the antibiotic-sensitive reference strain Ec ATCC25922 formed a substantial amount of biofilm at d 3 (A595nm of 2.207 ± 1.060). Large biomass variations were observed among biological replicates in the stronger biofilm-producer strains, particularly in the reference strain Ec ATCC25922. In this regard, many variables could affect biofilm production in microtiter plates as well as crystal violet assessment including slight variations in incubation times, incubation temperatures, little variations in the dye solution, and/or stochastic variations during the washing steps [28-30].Thus, depending on the type of biofilm and the strength of adherence that is present, some level of overestimation or underestimation of biofilm biomass might occur.

Fig 1

Biofilm formation ability of E. coli clinical strains.

Biofilms were developed in M9 medium onto polystyrene plates and biofilm biomass was determined by crystal violet staining (A595nm) after 1, 2 or 3 days. Values are means of at least three biological replicates, and error bars indicate standard deviations.

Nevertheless, based on the results obtained, the four strains selected to carry out further studies can be considered biofilm producers.

Minimum inhibitory concentration of 1,8-cineole

The minimum inhibitory concentration (MIC) of 1,8-cineole against the selected biofilm-producing E. coli strains were determined (Table 2). The MDR ESBL-producing strain Ec AM10 and the antibiotic-sensitive strain Ec ATCC25922 showed MIC values in the range of 0.5–2% (v/v) 1,8-cineole. A higher MIC was observed for the MDR ESBL-producing strain Ec 07 (MIC ≥2% of the phytochemical). The third MDR ESBL-producing strain under study, Ec AM7, was less susceptible to the phytochemical, showing only 14% inhibition of bacterial growth when 2% (v/v) 1,8-cineole was assayed.

Table 2

Minimum inhibitory concentration of 1,8-cineole against selected biofilm-producing E. coli strains.

Strain	1,8-cineole MIC range (%, v/v)
Ec ATCC25922	0.5–1
Ec AM7	>2a
Ec AM10	1–2
Ec 07	≥2

MIC, minimum inhibitory concentration. Values from biological triplicate experiments are shown.

a A 14% growth inhibition was reached with 2% (v/v) of the phytochemical.

Concentration-response and time-course effect of 1,8-cineole over cell viability in pre-formed biofilms

The ability of 1,8-cineole to affect cell viability in mature biofilms was analyzed. For this purpose, the strong biofilm-producer strain Ec AM7, that generates a substantial biofilm biomass at d 3, was chosen to determine the optimal treatment conditions. First, 3-d-old biofilms were challenged with increasing phytochemical concentrations (0.125 to 2%, v/v) or the corresponding amount of its vehicle ethanol (0.03 to 0.5%, v/v) during 1 h and viable cell counts were determined (Fig 2A and 2B). Fig 2A showed that the number of viable cells in biofilms was not modified by any of the ethanol concentrations tested. However, increasing concentrations of 1,8-cineole showed a concentration-dependent detrimental effect on bacterial viability (Fig 2B). A phytochemical concentration of 0.5% (v/v) caused a 1.5-log decrease in viable cell counts in the attached biofilm, whereas both 1 and 2% (v/v) 1,8-cineole showed the highest detrimental effect in biofilm viability (a 3-log decrease of viable cells). Time-course experiments in which biofilms were exposed to 1% (v/v) 1,8-cineole evidenced the highest cell viability reduction when the phytochemical was applied for 1 h, and no higher effect was observed 3 h after treatment (Fig 2C). Altogether, these results demonstrated the efficacy of a treatment with 1% (v/v) 1,8-cineole during 1 h to significantly diminish the number of viable cells in pre-formed E. coli biofilms.

Fig 2

Concentration-response and time-course effect of 1,8-cineole over cell viability in pre-formed E. coli biofilms.

Biofilms of the E. coli strain Ec AM7 developed for 3 d were challenged with increasing concentrations of the vehicle ethanol (A) or 1,8-cineole (B) for 1 h, and then the number of viable cells per cm2 were assessed after mechanically recover cells from polystyrene plates. In (B), values are means of five biological replicates. (#) p<0.05 compared to untreated control by one-way ANOVA followed by Bonferroni post-hoc test. (C) Three-days-old Ec AM7 biofilms were exposed to 1% (v/v) 1,8-cineole or the corresponding vehicle concentration (ethanol 0.25%, v/v) at different times, and the number of viable cells assessed as explained above. Values are means of four biological replicates, and error bars indicate standard deviations. (*) p<0.05 by Student´s t test.

Effect of 1,8-cineole over cell viability in biofilms of various MDR ESBL-producing E. coli clinical isolates

Next, the effect of this phytochemical treatment on cell viability in mature biofilms formed by the other E. coli strains under study was assayed (Fig 3). Significant reductions in the number of viable cells, ranging from 3- to 4-log, were observed in biofilms of all three tested bacteria, either sensitive to antibiotics or MDR ESBL-producers, compared to vehicle-treated controls. It should be noted that all tested strains were affected by 1% (v/v) 1,8-cineole in biofilms, independently of their susceptibility to the phytochemical when in planktonic state (MICs ranging from 0.5 to >2%, v/v). Altogether, these results clearly evidenced the antibiofilm activity of 1,8-cineole against pre-formed biofilms produced by both antibiotic-sensitive and MDR ESBL-producing strains of E. coli.

Fig 3

Effect of 1,8-cineole over cell viability of pre-formed E. coli biofilms.

Three-days-old biofilms of the E. coli strains Ec ATCC25922, Ec AM10, and Ec 07 were challenged with 1% (v/v) 1,8-cineole for 1 h, and then the number of viable cells per cm2 were assessed as described in legend of Fig 2. Values are means of three biological replicates, and error bars indicate standard deviations. (*) p<0.05 compared to vehicle by Student´s t test.

Evaluation of biofilm biomass disruption by 1,8-cineole treatment

The decrease in the number of viable cells in the biofilm observed after 1,8-cineole treatment could be caused by a disruption of the biofilm structure. To investigate this possibility, biofilm biomass was determined by crystal violet staining after phytochemical treatment (Table 3). Certain variations were observed in the remaining biomass detected between independent assays; these differences can be attributed to some mechanical disruption of biofilms produced during washing steps. Nevertheless, a clear reduction in biofilm biomass was observed in all four E. coli strain tested after 1 h treatment with 1% (v/v) 1,8-cineole, compared to their corresponding vehicle-treated controls (48–65% decrease in biofilm biomass). This result showed that, regardless of the amount of biofilm biomass produced by each strain, this compound was able to disrupt the biofilms in a similar percentage (more than 50% of biomass reduction in all tested strains).

Table 3

Effect of 1,8-cineole on pre-formed E. coli biofilms.

Strain	Biofilm biomass (A595nm)a		Biofilm disruption (%) (mean ± SD)
	Vehicle control (mean ± SD)	Treated with 1,8-C (mean ± SD)
Ec ATCC25922	1.76 ± 0.76	0.74 ± 0.44	60 ± 12 b
Ec AM7	2.66 ± 0.33	1.34 ± 0.37	48 ± 18 b
Ec AM10	0.73 ± 0.20	0.39 ± 0.25	49 ± 21 b
Ec 07	0.45 ± 0.12	0.16 ± 0.05	65 ± 02 b

a 3-d-old biofilms treated for 1 h with 0.25% (v/v) ethanol (vehicle control) or 1% (v/v) 1,8-cineole (1,8-C). Biological quadruplicates were performed.

b Significant difference (p < 0.05) compared to the vehicle control by Student´s t-test.

Assessment of bacterial viability of surface-attached cells after exposure to 1,8-cineole by confocal microscopic analysis

To evaluate whether 1,8-cineole is capable of killing E. coli Ec AM7 cells in biofilms, the biofilm samples were live/dead-stained for analysis by confocal laser scanning microscopy (CLSM). Fig 4A–4C presents the representative confocal images of the studies groups. Visualization of the biofilm structure in control (without any treatment) and vehicle-treated E. coli biofilms showed that the majority of cells were alive and only a few dispersed dead bacteria were observed (Fig 4A and 4B). In contrast, 1,8-cineole treated biofilms evidenced mostly dead cells and, remarkably, these dead bacteria were distributed throughout the biofilm structure (Fig 4C). Quantification of live and dead biomass by COMSTSAT quantitative analysis of confocal images indicated around 83% of viable bacteria in control biofilms, whereas 95% of cells in 1,8-cineole-treated biofilm were dead (Fig 4D).

Fig 4

Confocal laser scanning microscopy of LIVE/DEAD-stained E. coli biofilms.

Pre-formed biofilms (3 d-old) of the MDR ESBL-producing strain Ec AM7 were incubated for 1 h with (A) M9 medium (untreated), (B) 0.25% ethanol (vehicle), or (C) 1% (v/v) 1,8-cineole and were further incubated with the Live/Dead viability stain to show live (green) or dead (red/yellow) bacterial cells. Scale bars: 20 μm. (D) COMSTAT analysis of biomass. For each condition, the % of live and dead bacteria was calculated.

These results evidenced that biofilm treatment with 1,8-cineole during 1 h produced a high level of cell death within the biofilm.

Viability evaluation of biofilm-detached cells after 1,8-cineole treatment

As already shown here, 1,8-cineole treatment produced a significant loss of biofilm biomass, and consequent release of detached cells into the surrounding medium. It has been postulated that a good antibiofilm agent should not only attack bacteria into the biofilm but also display an action against biofilm-released cells [31]. To analyze this issue, viability of detached cells was assessed by two experimental approaches.

First, determination of cfu counting was performed (Fig 5A). In both untreated (medium alone) and vehicle-treated biofilms a substantial amount of viable bacteria were detected in the surrounding media (7.21×107 and 5.39×107 cfu/ml, respectively). This is likely due to the reported active dispersion of cells from mature biofilms [32], considering that a minimal planktonic growth would occur in this minimal medium in 1 h. On the other hand, the number of viable cells detached from phytochemical-treated biofilms was substantially lower (5.21×103 cfu/ml).

Fig 5

Viability of biofilm-detached cells after 1,8-cineole treatment.

Biofilm-detached cells from pre-formed biofilms (3 d-old) of the MDR ESBL-producing strain Ec AM7 were collected after 1 h incubation with medium alone (untreated), 0.25% ethanol (vehicle) or 1% (v/v) 1,8-cineole. (A) Determination of viable cells by cfu counting. Values are means of three biological replicates, and error bars indicate standard deviations. (*) p<0.01 compared to vehicle by Student´s t test. (B) Flow cytometry analysis after Live/Dead staining. Data were displayed as flow cytometric histograms of counted bacterial events (y-axis) associated cell fluorescence (x-axis). Marker M1 is the region that the damaged cells were stained by propidium iodide. For each sample, 105 cells were analyzed.

Second, detached cells were live/dead-stained for flow cytometry analysis (Fig 5B). As expected, a small proportion of cells released from vehicle-treated biofilms showed propidium iodide fluorescence signal (2.1% cells stained). Conversely, 1,8-cineole treatment caused a clear increase in propidium iodide fluorescence of the biofilm-detached cells (98.3% cells stained). This result indicated that the majority of the E. coli cells removed from the biofilm after the phytochemical treatment have their membrane integrity compromised.

Taken together, these results evidenced that after 1,8-cineole treatment, bacteria detached from E. coli biofilms were mostly dead cells.

Evaluation of biofilm regrowth after 1,8-cineole treatment

To investigate whether the biofilm cells surviving the 1,8-cineole treatment can grow to the level of before treatment, fresh medium was added to treated-biofilms and cell viability was assessed after 6 h and 24 h at 37°C (Table 4). Viable cells in control biofilms treated with vehicle were in the range of 2.10×107 to 5.90×107 cfu/cm2 in the time-period assayed. As observed earlier, 1 h exposure to 1% (v/v) 1,8-cineole (0 h post-treatment) diminished cell viability 3.5-log (to 5.80×103 cfu/cm2). After 6 h and 24 h in fresh medium, viable cell counts of phytochemical-treated biofilms increased to 3.70×104 and 1.60×105 cfu/cm2. These regrown biofilms showed at least 2.5-log lower cell counts than vehicle-treated biofilms.

Table 4

Regrowth of E. coli biofilms after 1,8-cineole treatment.

	Cell viability after treatment (Log10 cfu/cm2)a
	0 h post-treatment	6 h post-treatment	24 h post-treatment
Vehicle control	7.317 ± 0.042	7.316 ± 0.042	7.789 ± 0.109
Treated with 1,8-C	3.765 ± 0.273b	4.567 ± 0.064b	5.207 ± 0.039b

a 3-d-old biofilms treated for 1 h with 0.25% (v/v) ethanol (vehicle control) or 1% (v/v) 1,8-cineole (1,8-C) and then incubated in fresh M9 medium. Data correspond to mean ± SD of three biological replicates.

b Significant difference (p < 0.05) compared to the corresponding vehicle control by Student´s t-test.

The presented results evidenced the effectiveness of the 1,8-cineole treatment to limit biofilm regrowth.

Discussion

The extended biofilm recalcitrance toward antibiotic treatment has generated an urgent need for novel strategies against biofilm-associated infections [5]. We have previously reported that 1,8-cineole exhibits bactericidal activity against planktonic E. coli cells [12]. Therefore, we analyzed here the antibiofilm activity of this phytochemical against MDR ESBL-producing uropathogenic E. coli strains that are biofilm producers.

In the present study, the incidence of biofilm formation in MDR ESBL-producing uropathogenic E. coli was 30% (n = 10). Published studies have reported a great variability in biofilm-production ability by urine-associated E. coli strains, ranging for 13 to 69% of total strains studied (n = 100–250) [33-35]. These variations can be explained by intrinsic differences among individual E. coli isolates as well as variations in the experimental conditions used to assess biofilm formation. In this regard, it has been reported stronger biofilm formation in minimal media, such as M9, than in rich media by clinical strains of E. coli [36]. Here, we used M9 medium supplemented with glucose (0.8%) and the static model of biofilm formation on microtiter plate for biofilm formation assays. Even though the incidence of biofilm formation we found was moderated (30%), under a clinical point of view this result is relevant because biofilm-producing MDR bacteria not only increase the chronicity of urinary tract infection but also make the infection more recalcitrant to antibiotic treatment [5].Our new findings demonstrate that 1,8-cineole diminished the total number of viable cells in mature biofilms of a MDR ESBL-producing strain, in a concentration- and time-dependent manner. A bactericidal effect in biofilms was observed (viable cell reduction of 3–4 log) by applying during 1 h a concentration of 1% (v/v) 1,8-cineole (corresponding to a sub-MIC level). Mature biofilms formed by all E. coli strains tested in this study, both antibiotic-sensitive and MDR ESBL-producers, were susceptible to the phytochemical. Thus, the antibiofilm efficacy of 1,8-cineole reported here supports its use against E. coli strains forming relatively high biofilm biomasses.

As stated in the Introduction, the focus of this study was on the antibiofilm activity of 1,8-cineole against mature biofilms. At this stage, cells within the biofilm might be under stress from depleting nutrients and oxygen, and therefore this circumstance could impact the phytochemical's efficacy. In this regard, other researchers evidenced that the monoterpene carvacrol, a phytochemical with reported antimicrobial activity, was more biocidal during early biofilm development compared to mature biofilms formed by Staphylococcus aureus and Salmonella enterica [37]. Future work needs to be done to better understand the antibiofilm effect of 1,8-cineole over E. coli biofilms, particularly at an early developmental stage.

The observed decrease in the number of viable biofilm-forming cells after 1,8-cineole treatment could be attributed either to a disruption of the biofilm structure or to a direct bactericidal effect in the biofilm. Concerning the first possibility, the compound was able to decrease the biomass of pre-formed biofilms by approximately 50%. Moreover, the majority of the detached cells were found dead by flow cytometric analysis. Regarding the second hypothesis, we visualized by confocal microscopy that most of the adherent cells remaining in biofilms after 1,8-cineole challenge were dead, as judged by uptake of the normally membrane-impermeant dye propidium iodide.

The regrowth of biofilms after antimicrobial treatments has been considered as a critical reason for the persistent biofilm infection [38]. Our findings indicate that biofilm regrowth after 1,8-cineole treatment is limited, as 24 h after treatment there was still a 2.5 logs lower cell counts than in control biofilms.

From a clinical point of view, monotherapy have limited efficacy in the treatment of urinary tract infections caused by MDR ESBL-producing E. coli and combination of antimicrobial agents may be of clinical interest [39]. In this regard, as 1,8-cineole killed the majority of, but not all, E. coli cells forming the biofilm, further investigation focused on possible synergistic interactions of this phytochemical with common antibiotics would have important clinical implications for the treatment of biofilm-related infections involving E. coli.

The antimicrobial activities of individual compounds that are main constituents of plant volatile oils have been extensively studied in planktonic bacteria, however, relatively few of them have been investigated against biofilms formed by uropathogenic E. coli. Phytochemicals such as cinnamaldehyde from cinnamon oil [40], carvacrol from oregano oil [41], and thymol from thyme red oil [41,42] have been reported to reduce E. coli biofilm formation at sub-MIC concentrations. Nevertheless, none of these studies analyzed whether the compounds were able to disrupt pre-established biofilms since they were added at the beginning of the experiment.

A number of studies have reported both MIC and bactericidal effect of 1,8-cineole against planktonic E. coli to be in the range of 0.25–6.25% (v/v) [12,13,15,43,44]. However, there are few reports in the literature where 1,8-cineole has been tested as an anti-biofilm agent against E. coli. In [13] the authors studied the antimicrobial efficacy of this phytochemical against an antibiotic-sensitive E. coli strain. In this report, a bactericidal effect on a pre-established biofilm (24 h-old) was observed after 24 h exposure to 256 g/l (27.8% v/v) 1,8-cineole, concentration corresponding to 4-times the MIC (MIC = 64 g/l, 6.25% v/v). Recently, it has been reported a 50% biomass reduction of a preformed (18 h-old) E. coli biofilm by applying a sub-MIC concentration of 1,8-cineole during 24 h [45]. However, no information regarding cell viability in those treated biofilms was provided.

Our work here clearly shows that 1 h challenge with 1,8-cineole caused both biomass reduction and cell death in pre-formed biofilms of MDR ESBL-producing uropathogenic E. coli isolates, at concentrations that were not lethal for planktonic cells. Thus, we obtained an effective anti-biofilm effect on mature E. coli biofilms by applying a sub-MIC phytochemical concentration for a short period of time (1h). The reasons behind the discrepancy between our findings and other laboratory´s results are not yet fully understood. Differences in the bacterial strains used, the experimental conditions for biofilm development (type and size of the surface, culture medium, biofilm age), and treatment duration might impact the final outcome.

Although the exact mechanism behind the antibiofilm effect of 1,8-cineole against uropathogenic E. coli is not yet entirely known, our findings demonstrate that the phytochemical is able to partially disrupt the biofilm, as well as to directly kill bacteria within the biofilm. Notably, our confocal images revealed that the plant compound affected the entire biofilm, including not only the outermost layer but also the innermost cells of the biofilm. This behaviour is in accordance with the idea that small non-polar components of plant volatile oils, having a superior diffusion coefficient than common antibiotics, present a high biofilm penetration potential [46]. The antibiofilm activity of 1,8-cineole can be attributed, at least in part, to membrane permeabilization of biofilm-forming cells upon penetration into the biofilm structure, as this monoterpene exhibit a bactericidal activity against planktonic E. coli cells that is associated with injury to the cell membrane [12]. Other authors have reported Staphylococcus aureus biofilm inhibition by 1,8-cineole, in the context of a chronic rhinosinusitis model, that was correlated with a decrease of proliferation and a down-regulation of major key players in biofilm generation (agrA, SarA and σB genes) [45].

The management of urinary tract infections has become more difficult because of the increasing prevalence of MDR strains and the inability of antibiotics to fully eradicate biofilm-embedded bacteria. Altogether, our findings suggest that1,8-cineole exhibit an outstanding advantage in terms of agent accessibility to biofilm-based infectious diseases, overcoming tolerance antimicrobial mechanisms and causing cell death and biomass reduction in biofilms formed by MDR ESBL-producing uropathogenic E. coli strains.

Conclusions

This study is the first to demonstrate the antibiofilm activity of 1,8-cineole against MDR ESBL-producing E. coli. Notably, the compound is able to cause substantial bacterial dead into the biofilm-attached and biofilm-released cells. Therefore, we propose this phytochemical as a potential compound in development of novel E. coli antibiofilm agents.

1 Apr 2020

PONE-D-20-04063

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Vazquez and colleagues present a manuscript that highlights an important issue - antibiotic resistance. With growing resistance to currently available therapeutics - natural compounds are of growing interest as potential biofilm inhibitors.

The manuscript is well written - however I have some concerns about the methodology used and the associated explanation for the chosen methods.

- Why did the authors choose a 3 day biofilm assay? Response to antimicrobial strategies in bacterial populations can be observed at the transcript level (RNA) after ~30 minutes treatment, by growing biofilms for 3 days - my concern at this small scale is that cells within the biofilm are under stress from depleting nutrients and oxygen, and therefore will impact any live/dead assessment being made for the inhibitor.

A time course with treatment would be a good way to test how the inhibitor's efficacy is impacted by depleting biofilm health.

-Were flow experiments only performed on dispersed cells? These are likely to be live as they are active - flow should be performed on biofilm cells also to calculate quantity of live/dead. This was not clear in the manuscript.

- 1,8 - cineole may be acting as a dispersal agent - what are the potential health consequences of this in a patient? This should be discussed by the authors. Also additional dispersal assays would strengthen the manuscript. Is motility involved?

Reviewer #2: This paper's major issue is that the English is not of sufficient quality to review. I believe that the experiments conducted and the methods used are likely sufficient to address the question but at this time the paper must be largely re-written. Due to this I have been unable to fully review the paper but this was my review of the parts I could review.

I dislike the term essential oil in a scientific paper. It does not convey a clear description of what the product is. Clearer terms such as plant oil. It can also be misappropriated by alternative medicine proponents. The authors are I assume not working under the guise that the aroma of the oil is in anyway contributing to the antibacterial nature of the oil. If that hold true then the use of ‘essential oil’ should be avoided.

In terms of scientific questions, the authors layout the following approach. Using the plant extracted oil a substantial reduction in viable cells was observed (a 3-fold log reduction). This was result was obtained in both antibiotic resistant and sensitive cells. This is then expanded to say that there is a reduction in biomass. The remaining biomass was then subjected to confocal analysis of live/dead staining and showed part of the remaining adhered biomass was actually dead. Cell no longer adhered we found to be mostly dead by flow cytometry analysis.

The use of statistics seems reudementary but without access to the raw data (as required by PLOS one) it is hard to determine. I would argue that given your hypothesis and previous work a two tailed test is not correct, you are working under the hypothesis that the plant extract will reduce the biofilm and or cell viability.

Regards

James Gurney

**********

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Reviewer #2: Yes: James Gurney

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6 May 2020

May 06, 2020

Dear Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Please find enclosed our revised version of the manuscript number PONE-D-20-04063 entitled “Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole” for your consideration. It has been revised and changes have been made for each specific comment of the reviewers, as addressed below (modifications are indicated, referring to line numbers in the Marked Up Manuscript file).

Reviewer #1

General comment: Vazquez and colleagues present a manuscript that highlights an important issue - antibiotic resistance. With growing resistance to currently available therapeutics - natural compounds are of growing interest as potential biofilm inhibitors.

The manuscript is well written - however I have some concerns about the methodology used and the associated explanation for the chosen methods.

Author response to general comment: We now better explain the rational of the methodology used (see L248-251; L316-318; L324-327; L340-349; L353-354).

Specific comment #1: Why did the authors choose a 3 day biofilm assay? Response to antimicrobial strategies in bacterial populations can be observed at the transcript level (RNA) after ~30 minutes treatment, by growing biofilms for 3 days - my concern at this small scale is that cells within the biofilm are under stress from depleting nutrients and oxygen, and therefore will impact any live/dead assessment being made for the inhibitor.

A time course with treatment would be a good way to test how the inhibitor's efficacy is impacted by depleting biofilm health.

Author response #1: We now better explain in the text the reason for choosing a 3 day-old biofilm (to assess the phytochemical antibiofilm activity over a mature robust biofilm with a substantial biomass, as showed by crystal violet assay) (see L248-250 and L278).

We agree with the reviewer´s comment regarding that cells within 3-d-old biofilms could be under stress for depleting nutrients and oxygen. We now discuss the possibility that the inhibitor´s efficacy could be impacted by this situation and, therefore, that future work is needed to analyze this possibility (see L400-403, L406-408). In addition, results reported by other group, that applied the phytochemical carvacrol at different stages of biofilm development, has been included in the Discussion (see L403-406). These authors found that the compound was more biocidal during early biofilm development compared to mature biofilms.

Specific comment #2: Were flow experiments only performed on dispersed cells? These are likely to be live as they are active - flow should be performed on biofilm cells also to calculate quantity of live/dead. This was not clear in the manuscript.

Author response #2: We now better explain the rational for the experiments to analyse cell viability after 1,8-c treatment on cells detached from the biofilms (by cfu counting and live/dead staining followed by flow cytometry) (see L339-363). On the other hand, we now re-write the paragraph corresponding to assessment of bacterial viability of surface-attached cells by confocal microscopy, to clearly explain that quantification of live/dead cells into biofilms was performed by confocal microscopy followed by COMSTSAT quantitative analysis. (see L316-327).

Specific comment #3- 1,8 - cineole may be acting as a dispersal agent - what are the potential health consequences of this in a patient? This should be discussed by the authors. Also additional dispersal assays would strengthen the manuscript. Is motility involved?

Author response #3: We now better explain the effect of 1,8-cineole as partially disrupting the biofilm and causing detachment of mainly dead cells (see L340-364). Nevertheless, because we detected a minor amount of detached cells still alive, we now discuss the importance to further investigate the possible synergistic interactions of 1,8-cineole with common antibiotics to make the phytochemical treatment more efficient in a patient (see L424-430). Besides, we believe that further exploration of the biofilm detachment events caused by the phytochemical is beyond the scope of the present work.

Reviewer #2

General comments: This paper's major issue is that the English is not of sufficient quality to review. I believe that the experiments conducted and the methods used are likely sufficient to address the question but at this time the paper must be largely re-written. Due to this I have been unable to fully review the paper but this was my review of the parts I could review.

Author response to general comment: We revised the whole manuscript for proper English usage, several parts of the text has been re-written and we consider that its quality has now been improved.

Specific comment #1: I dislike the term essential oil in a scientific paper. It does not convey a clear description of what the product is. Clearer terms such as plant oil. It can also be misappropriated by alternative medicine proponents. The authors are I assume not working under the guise that the aroma of the oil is in anyway contributing to the antibacterial nature of the oil. If that hold true then the use of ‘essential oil’ should be avoided.

Author response #1: As suggested, the term “essential oil” has been changed by “plant volatile oil” or “volatile oil” all along the manuscript ( see L28,83,87,90,431,453).

Specific comment #2: In terms of scientific questions, the authors layout the following approach. Using the plant extracted oil a substantial reduction in viable cells was observed (a 3-fold log reduction). This was result was obtained in both antibiotic resistant and sensitive cells. This is then expanded to say that there is a reduction in biomass. The remaining biomass was then subjected to confocal analysis of live/dead staining and showed part of the remaining adhered biomass was actually dead. Cell no longer adhered we found to be mostly dead by flow cytometry analysis.

The use of statistics seems reudementary but without access to the raw data (as required by PLOS one) it is hard to determine. I would argue that given your hypothesis and previous work a two tailed test is not correct, you are working under the hypothesis that the plant extract will reduce the biofilm and or cell viability.

Author response #2: As the reviewer requested, we now provide access to all the raw data through the Figshare repository (https://doi.org/10.6084/m9.figshare.c.4964399.v1). According with the reviewer´s comment, we revised the statistics used and now a one-tailed Student´s t-test was applied to our data (see L202 and test results showed in the repository).

We thank the reviewers and editors for their constructive comments and suggestions. We believe that the revised manuscript is now acceptable for publication in PlosOne.

Sincerely yours,

Estela Galván

Submitted filename: Galvan R1 - Response to Reviewers.docx

Click here for additional data file.

9 Jun 2020

PONE-D-20-04063R1

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

PLOS ONE

Dear Dr. Galvan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 24 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Second review of the proposed plos one article

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

The authors have made several key improvements to the English as evident by the tracked changes version, however there are a number of flaws still remaining that will need addressing. Plos One does not copy edit so they must be fixed before acceptance.

The improved English has allowed me to complete a full review. I have 4 major concerns and a number of minor concerns; I will include the errors in English under the minor concerns where I have spotted them, I will not correct them only point out where I believe there is a flaw.

Major concerns

How much of the reduction is due to the presence of tween 80? Previous work using the same compound did not make use of the surfactant. Why was it used and why was it not controlled for? On line 219 the authors make mention of reduced bacterial growth. They should present this data. Did they do a tween negative control? If not, they should. Line 389-392: has to be expanded significantly, currently other published work shows a much higher level for both MIC and bactericidal of the oil. What rational do the authors have for this discrepancy?

Given that the majority of clinical isolates did not make biofilms the authors must address the rational for using a biofilm disrupting treatment. I see three possibilities, and all should be discussed. Either the medium (M9) is not sufficient to recapitulate the clinical environment, reducing the enthusiasm for this work. Or the presence of biofilm forming strains in actual clinical samples is limited raising question about the usefulness of this treatment. Third, perhaps samples collected from clinical are bias for not biofilm forming as they would presumably be easier to collect. I would ask the author to at least address point 1 and 2 and remove all work related to the strain that did not produce biofilms.

The authors use 3 different methods of approximating biofilms. Crystal violet, CFUs, and confocal live dead staining. All 3 methods gave wildly different levels of reduction. For example, why did the CFU have 3-4 log reduction while the confocal had around a 1-2 log fold? Why don’t these methods agree? This I find further concerning as the MIC does not agree with the biofilm data. I would revise the section from line 364-379 to address this problem.

Section starting on line 309. Are the cells actually released from the biofilm or are they just growing planktonically? If they are released than according to fig 5A the ethanol treatment is better at releasing cells. If they are just growing planktonically this section needs to be revised.

Minor

Line 35: English

Line 38: English

Line 63: Needs a citation

Line 68: needs a citation

Line 74: why differentiate between fungi and yeast?

Line 83-84 I would remove the QS section. It adds nothing.

Line 116: English in two instances. Freshly streaked doesn’t mean anything and you do not streak *in* agar but *on* it.

Line 117: what is the volume used for the overnights?

Line 136: Why did you only use duplicates here? This data is not robust for statistically analysis. Please state all replicate numbers in figure legends

Line 146: space between tween 80 is missing.

Line 149: state what the vehicle is and how much was used.

Line 152: was only 1 assay done as the sentence implies? Or should it be biofilm biomass and cell viability?

Line 155: English

Line 182: English

Line 216: table say equal or greater to, but text suggests it is sensitive to 2%

Line 236: English

Line 248: and other parts, the authors say at least 3 replicates, are there uneven tests? What did the authors do to reduce the P-hacking of sampling at different rates per treatment? Or is this a case of an English mistake?

Line 254-255: Why does biomass (Fig 1) not correlate with figure 3?

Line 260: English

Line 282: What is the replicate number for data in table 3

Line 288 English

Line 316 & 318: give exact numbers.

Line 366: English

Line 411: English

Reviewer #3: General comments. Vazquez and colleagues present a manuscript evaluating the antibiofilm activity of 1,8-cineole against pre-formed mature biofilms of uropathogenic multidrug-resistant E. coli clinical strains.

The manuscript is well written and experiments are presented on a rational base.

However I have the following concerns:

Specific comment #1. The authors evaluate biofilm formation of ten E. coli isolates. Only one of them presented a substantial biofilm formation and two a mild formation. Raw data presented supports these conclusions. However data dispersion of biofilm formation in biofilm-forming strains is unusual. Authors should discuss these anomalies.

Specific comment #2. Authors should analyze the low frequency of biofilm-forming isolates in the context of evaluating a possible biofilm inhibition treatment.

Specific comment #3. Fig2A. The authors show cell viability on biofilms when they increased 1,8 cineole concentration. In raw data authors present five assays for treatment experiment but only one for control. Although differences between treatment and control with concentrations > 0.5% are important, authors must demonstrated this with a test. I don´t think the curve representation is the best suitable for these result.

Authors can consider present results in two parts: ethanol in extracts is not detrimental for survival and on the other side, dependence of viability to 1,8 cineole concentration compared to non 1,8 cineole (first column on raw data).

Specific comment #4. Table 3. The dispersion values between assays are noteworthy. Although differences are significant authors should address why they have such differences between assays. Dispersion was not observed in raw data for fig 1. I assume that some disruption of biofilm was produced during washes during treatment.

Specific comment #5. Authors described viability of detached cells arguing “It has been postulated that a good antibiofilm agent should not only attack bacteria into the biofilm but also display an action against biofilm-released cells”. The results is in concordance with high rate of dead cells on biofilm. It would be more interesting and will significantly improve the impact of the work to investigate if a post-treatment biofilm is able to growth.

Specific comment #6. Authors did not demonstrate that high biofilm biomasses yielded by strains are consequence of thicker extracellular matrix. Statement in L388-L393 should be modified.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: James Gurney

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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20 Jul 2020

Estela M. Galvan, Ph D

Lab. of Bacterial Pathogenesis

Centro de Estudios Biomedicos, Biotecnologicos,

Ambientales y Diagnostico (CEBBAD)

Universidad Maimonides

Hidalgo 775

C1405BWE-Buenos Aires, Argentina

July 20, 2020

Dear Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Please find enclosed our revised version of the manuscript number PONE-D-20-04063R1 entitled “Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole” for your consideration. It has been revised and changes have been made for each specific comment of the reviewers, as addressed below (modifications are indicated, referring to line numbers in the Marked Up Manuscript file).

Reviewer #2: Second review of the proposed plos one article

General comment: The authors have made several key improvements to the English as evident by the tracked changes version, however there are a number of flaws still remaining that will need addressing. Plos One does not copy edit so they must be fixed before acceptance.

Author response to general comment: We addressed all marked flaws. We consider that now the whole manuscript has proper English usage.

Major concern #1: How much of the reduction is due to the presence of tween 80? Previous work using the same compound did not make use of the surfactant. Why was it used and why was it not controlled for?

On line 219 the authors make mention of reduced bacterial growth. They should present this data. Did they do a tween negative control? If not, they should.

Line 389-392: has to be expanded significantly, currently other published work shows a much higher level for both MIC and bactericidal of the oil. What rational do the authors have for this discrepancy?

Author response #1: Tween 80, as well as DMSO, are commonly used to increase solubility of lipophilic molecules, such as essential oils and their individual compounds, in aqueous solutions (Man and Markham, 1998; Ojeda-Sana et al., 2013; Kwiatkowski et al., 2019). The effect of Tween 80 (0.5 %) was tested, and it did not show any detrimental effect neither on planktonic growth (MIC determinations) nor on biofilms (biomass, cell viability, cell detachment). We have now rewritten Material and methods section to clarify this issue (see L 143-154 and L164-170).

Regarding the reduction of bacterial growth mentioned on line 219, we now presented the data in Table 2 (see L253). As explained above, tween negative controls have been performed.

As requested, additional published work regarding MIC and bactericidal effect of 1,8-cineole has been included and discussed in the context of our findings (see L457-479).

Major concern #2: Given that the majority of clinical isolates did not make biofilms the authors must address the rational for using a biofilm disrupting treatment. I see three possibilities, and all should be discussed. Either the medium (M9) is not sufficient to recapitulate the clinical environment, reducing the enthusiasm for this work. Or the presence of biofilm forming strains in actual clinical samples is limited raising question about the usefulness of this treatment. Third, perhaps samples collected from clinical are bias for not biofilm forming as they would presumably be easier to collect. I would ask the author to at least address point 1 and 2 and remove all work related to the strain that did not produce biofilms.

Author response #2: We addressed this reviewer’s concern by adding a paragraph in the Discussion section (see L395-407). Additionally, strains that did not produce biofilm were removed from Fig 1.

Major concern #3- The authors use 3 different methods of approximating biofilms. Crystal violet, CFUs, and confocal live dead staining. All 3 methods gave wildly different levels of reduction. For example, why did the CFU have 3-4 log reduction while the confocal had around a 1-2 log fold? Why don’t these methods agree? This I find further concerning as the MIC does not agree with the biofilm data. I would revise the section from line 364-379 to address this problem.

Author response #3: For Ec AM7 biofilms, our results showed 2-4 log cell viability reduction by CFU counts and ⁓ 2 log reduction by confocal live/dead microscopy. These results are within the inter-experimental variation observed. In addition, while CFU counting assesses viable cells in the whole biofilm sample, confocal microscopy explores a limited number of microscopic fields of view.

Regarding the reviewer´s concern that MIC does not agree with the biofilm data, we consider that this is an important finding of the present work. Notably, the phytochemical showed better antimicrobial effect on biofilms compared to planktonic cells. This is in agreement with a recent report (Schurmman et al, 2019). We now better discuss this issue in the manuscript (see L465-475).

Major concern #4- Section starting on line 309. Are the cells actually released from the biofilm or are they just growing planktonically? If they are released than according to fig 5A the ethanol treatment is better at releasing cells. If they are just growing planktonically this section needs to be revised.

Author response #4: To address this reviewer´s comment we now included additional experimental data in Fig 5A. We also added a paragraph in the Results section regarding this issue (see L360-364 and L380).

Minor concerns

#1. Line 35: English. Response: Modified (see L35).

#2. Line 38: English. Response: Modified (see L39).

#3. Line 63: Needs a citation. Response: As requested, a citation has now been included (see L63).

#4. Line 68: needs a citation. Response: As requested, a citation has now been included (see L68).

#5. Line 74: why differentiate between fungi and yeast? Response: We regret this mistake and now the sentence was re-written (see L74).

#6. Line 83-84 I would remove the QS section. It adds nothing. Response: As requested, this sentence has been removed (see L83-84).

#7. Line 116: English in two instances. Freshly streaked doesn’t mean anything and you do not streak *in* agar but *on* it. Response: This sentence has been now modified (see L116).

#8. Line 117: what is the volume used for the overnights? Response: The requested information has now been added (see L117).

#9. Line 136: Why did you only use duplicates here? This data is not robust for statistically analysis. Please state all replicate numbers in figure legends. Response: Experiments were done in biological triplicate and technical duplicates were done. We now clarified this information in Materials and Methods section (see L129-131, L138-139). This data is robust for statistical analysis, as detailed in the raw data. All biological replicate numbers are stated in figure legends.

#10. Line 146: space between tween 80 is missing. Response: Corrected (see L162).

#11. Line 149: state what the vehicle is and how much was used. Response: Modified as requested (see L164-170).

#12. Line 152: was only 1 assay done as the sentence implies? Or should it be biofilm biomass and cell viability? Response: This sentence has been corrected (see L171).

#13. Line 155: English. Response: Modified (see L175).

#14. Line 182: English. Response: Modified (see L202-203).

#15. Line 216: table say equal or greater to, but text suggests it is sensitive to 2%. Response: For clarity, the corresponding sentence was re-written (see L241-245).

#16. Line 236: English. Response: Modified (see L270-274).

#17. Line 248: and other parts, the authors say at least 3 replicates, are there uneven tests? What did the authors do to reduce the P-hacking of sampling at different rates per treatment? Or is this a case of an English mistake? Response: We apologize for any English mistake. We now re-write these sentences to better explain what has been done (see L283, L288-289, L307-308, L327, L383).

#18. Line 254-255: Why does biomass (Fig 1) not correlate with figure 3? Response: Fig 1 shows biofilm biomass assessed by crystal violet whereas fig 3 shows cell viability of biofilms assessed by colony forming units (CFU). Whereas the crystal violet dye detects negatively-charged molecules in the biofilm (such as polysaccharides in the biofilm matrix, and lipopolisaccharides from bacterial cells), the CFU counting is the standard technique to detect viable cells. Because each technique detects different characteristics of the biofilm, no strict correlation between them is expected. Anyhow, for example, biofilm biomass and CFU numbers were higher for Ec ATCC25922 biofilms than for Ec AM10 biofilms.

#19. Line 260: English. Response: Modified (see L301-302).

#20. Line 282: What is the replicate number for data in table 3. Response: The requested information has now been added (see L327).

#21. Line 288 English. Response: Modified (see L332-333).

#22. Line 316 & 318: give exact numbers. Response: Modified as requested (see L362).

#23. Line 366: English. Response: Modified (see L430).

#24. Line 411: English. Response: Modified (see L502).

Reviewer #3

Specific comment #1: The authors evaluate biofilm formation of ten E. coli isolates. Only one of them presented a substantial biofilm formation and two a mild formation. Raw data presented supports these conclusions. However, data dispersion of biofilm formation in biofilm-forming strains is unusual. Authors should discuss these anomalies.

Author response #1: As requested, we now discuss this issue in the manuscript (see L227-230).

Specific comment #2: Authors should analyze the low frequency of biofilm-forming isolates in the context of

evaluating a possible biofilm inhibition treatment.

Author response #2: As requested, we now discuss this findings (see L396-408).

Specific comment #3: Fig2A. The authors show cell viability on biofilms when they increased 1,8 cineole

concentration. In raw data authors present five assays for treatment experiment but only one for control. Although differences between treatment and control with concentrations > 0.5% are important, authors must demonstrate this with a test. I don´t think the curve representation is the best suitable for these result.

Authors can consider present results in two parts: ethanol in extracts is not detrimental for survival and on the other side, dependence of viability to 1,8 cineole concentration compared to non 1,8 cineole (first column on raw data).

Author response #3: As requested, the concentration-dependent data in the old Fig. 2A is now displayed in two parts: ethanol effect in one panel (A) and 1,8-cineole effect in other panel (B). We additionally performed an ANOVA test to analyze significance of differences in cell viability after treatment (see new Fig2).

Specific comment #4: Table 3. The dispersion values between assays are noteworthy. Although differences are

significant authors should address why they have such differences between assays. Dispersion was not observed in raw data for fig 1. I assume that some disruption of biofilm was produced during washes during treatment.

Author response #4: We have now included a sentence to discuss variations between assays (see L316-318).

Specific comment #5: Authors described viability of detached cells arguing “It has been postulated that a good

antibiofilm agent should not only attack bacteria into the biofilm but also display an action against biofilm-released cells”. The results is in concordance with high rate of dead cells on biofilm. It would be more interesting and will significantly improve the impact of the work to investigate if a post-treatment biofilm is able to growth.

Author response #5: We agree with the reviewer´s comment regarding post-treatment biofilms. Even though this experiment is beyond the scope of the present work, we now mention in the manuscript their importance (see L435-436).

Specific comment #6: Authors did not demonstrate that high biofilm biomasses yielded by strains are consequence of thicker extracellular matrix. Statement in L388-L393 should be modified.

Author response #6: The requested statement has been modified (see L415-416).

We thank the reviewers and editors for their constructive comments and suggestions. We believe that the revised manuscript is now acceptable for publication in PlosOne.

Sincerely yours,

Estela Galván

Submitted filename: Galvan R2 - Response to Reviewers.docx

Click here for additional data file.

12 Aug 2020

PONE-D-20-04063R2

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

PLOS ONE

Dear Dr. Galvan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 26 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: Although most of the comments have been addressed, there are still a number of flaws that need to be addressed.

# one

The authors cite an article by Kragh et al to justify the stochastic variation in the biofilm results for the 72-hour biofilm and discourage the use of the CV staining method to draw conclusions. However, variations in biomass quantification were observed by microscopy (CV results were not shown) and were not as large as presented in this manuscript.

Authors must discuss the comment more extensively than citing the article by Kragh et al.

# two

Specific comment # 5. I disagree with the authors. I think the suggested experiment would improve significantly

impact of work. The authors present a new drug capable of altering and killing the cells presented in the biofilm life form. It is important to determine if the cells can grow.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: James Gurney

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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24 Sep 2020

Estela M. Galvan, Ph D

Lab. of Bacterial Pathogenesis

Centro de Estudios Biomedicos, Biotecnologicos,

Ambientales y Diagnostico (CEBBAD)

Universidad Maimonides

Hidalgo 775

C1405BWE-Buenos Aires, Argentina

September 24, 2020

Dear Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Please find enclosed our revised version of the manuscript number PONE-D-20-04063R2 entitled “Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole” for your consideration. It has been revised and changes have been made for each specific comment of the reviewers, as addressed below (modifications are indicated, referring to line numbers in the Marked Up Manuscript file).

Reviewer #3:

General comment: Although most of the comments have been addressed, there are still a number of flaws that need to be addressed.

Concern #1: The authors cite an article by Kragh et al to justify the stochastic variation in the biofilm results for the 72-hour biofilm and discourage the use of the CV staining method to draw conclusions. However, variations in biomass quantification were observed by microscopy (CV results were not shown) and were not as large as presented in this manuscript. Authors must discuss the comment more extensively than citing the article by Kragh et al.

Author response #1: As requested, we have now discuss more extensively the experimental variables that could account for the large biomass variations observed among biological replicates in the stronger biofilm-producer strains, particularly in the reference strain Ec ATCC25922 (see L231-242).

Concern #2: Specific comment # 5. I disagree with the authors. I think the suggested experiment would improve significantly impact of work. The authors present a new drug capable of altering and killing the cells presented in the biofilm life form. It is important to determine if the cells can grow.

Author response #2: As requested, new experimental data related to whether a post-treatment biofilm is able to regrow have been added (see L41-42, L169-176, L391-409 –including new Table 4-, L453-458).

We thank the reviewers and editors for their constructive comments and suggestions. We believe that the revised manuscript is now acceptable for publication in PlosOne.

Sincerely yours,

Estela Galván

Submitted filename: GalvanR3 -Response to Reviewers.docx

Click here for additional data file.

26 Oct 2020

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

PONE-D-20-04063R3

Dear Dr. Galvan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: While I signed off on the last version of this manuscript, I agree with reviewer 3 that the requested additions have strengthened the paper. My old comment is that I think the regrowth control should not have been a culture that had reach carrying capacity. Instead, I would have preferred to see the authors disrupt and dilute the biofilm so that the CFU was close to the treated level and watch for regrowth. However given the clear results of lower regrowth, I think the current assay is sufficient.

James Gurney

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: James Gurney

Reviewer #3: No

28 Oct 2020

PONE-D-20-04063R3

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

Dear Dr. Galván:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Monica Cartelle Gestal

Academic Editor

PLOS ONE