Journal of Antimicrobial Chemotherapy

Gupta, Preeti;Mankere, Bharti;Keloth, Shami Chekkoora;Tuteja, Urmil;Pandey, Pratibha;Chelvam, Kulanthaivel Thava

2018 Journal of Antimicrobial Chemotherapy

doi: 10.1093/jac/dky127pmid: 29688490

Abstract Background Vibrio cholerae, the aetiological agent of the deadly diarrhoeal disease cholera, is known to form biofilm. The antibiotic susceptibility status of biofilm of V. cholerae O139, an important epidemic strain in India and other countries, has not previously been studied in detail. Methods Antibiotic susceptibility status of planktonic and biofilm cultures of V. cholerae O139 was evaluated by determining MIC, MBC and minimum biofilm eradication concentration (MBEC) values of five different classes of antibiotics using established methods. Effects of antibiotic treatment on planktonic and biofilm cultures were analysed by scanning electron microscopy. The virulence of the antibiotic-surviving population (ASP) was evaluated using an infant mouse model. The frequency of spontaneous mutants and inheritability of antibiotic resistance were determined with standard methods. Results The antibiotic resistance exhibited by biofilm of V. cholerae O139 was found to be significantly higher (P < 0.05) than its planktonic counterpart. The biofilm-associated antibiotic resistance was found to be transient and exclusive to the biofilm culture. The frequency of ASP clones among antibiotic-treated biofilm cultures occurred at a rate of 0.012%–0.95% and these clones were found to retain the virulence and antibiotic resistance of their parent strains. Conclusions The biofilm of V. cholerae O139 was found to be resistant to different types of antibiotics tested. This unconventional biofilm resistance highlights the hidden danger of antimicrobial escape by V. cholerae, increased risk of cholera transmission and its continued persistence in the environment. Introduction Cholera remains a major healthcare burden in endemic areas with an estimated 2.9 million cases annually.1 Treatment options for this highly incapacitating disease rely mainly on prompt and proper rehydration therapy.2 However, the severe form of this disease requires treatment with antibiotics for rapid recovery. The severity of the illness, in terms of the duration and volume of diarrhoeal discharge, has been shown to be reduced with antibiotic treatment,3,4 which is crucial for patient care and judicious use of resources in developing countries. Even though antibiotic treatment has been shown to be associated with successful clinical outcomes, follow-up studies with cholera patients have documented failures in bacteriological clearance,5 raising concerns regarding the prime goal of antimicrobial therapy, which aims to achieve complete eradication of the aetiological agent.6 It has been postulated that in vivo-formed biofilm of Vibrio cholerae contributes to enhanced infectivity and environmental persistence.7–10 If this hypothesis were true in the clinical scenario, the bacteriological failure reported in the follow-up studies5,11 might be linked to the in vivo-formed biofilm, although this requires further study. Like other bacteria, V. cholerae has also been known to form biofilm under hostile conditions.12,13 A worrisome fact is that the biofilm form of many chronic and nosocomial pathogens has been shown to be much more resistant to antibiotics compared with their respective planktonic form.14,15 As of now, the clinical importance of antibiotic-resistant biofilm is registered mainly with chronic infections. However, cholera is an acute infection and the importance of its biofilm with respect to the clinical scenario is grossly ignored. Nevertheless, if the postulation of biofilm formation in the intestinal environment7–10,16 happens to be true, the immediate question that arises is about the susceptibility status of this biofilm form to the antibiotics administrated during treatment. In this line of interest, as we could find no detailed study in the literature, we aimed to address this gap with our present work because it would be noteworthy to understand the attributes that govern the antimicrobial escape by V. cholerae in the actual scenario that eventually leads to treatment failure and spread of cholera through patient stools. Materials and methods Bacterial strains and antibiotics V. cholerae O139 strain MO10 (MTCC 3906) was obtained from the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. It is resistant to trimethoprim, co-trimoxazole and streptomycin. Escherichia coli (ATCC 25922) was used as a control strain for antibiotic susceptibility tests. Antibiotics, namely ampicillin, ciprofloxacin, ceftriaxone, doxycycline, erythromycin and azithromycin (Sigma–Aldrich, Bangalore, India), in the range of 0.0156–2048 mg/L, were prepared in Mueller–Hinton (MH) broth, pH 7.2 (Hi-Media) for susceptibility tests. In vitro biofilm assay V. cholerae O139 biofilm was prepared using 96-well microtitre plates for 8, 12, 24, 36, 48 and 72 h, as described by Ceri et al.17 Biofilm quantification and the cell density at different timepoints were determined by the crystal violet assay18 and viable cell counts, respectively. For antibiotic degradation and penetration assays, 10 μL of diluted (1:100) overnight grown culture of V. cholerae O139 was deposited on a 0.2 μm nitrocellulose membrane, which was placed on an LB plate and incubated for 48 h at 30°C.19 For scanning electron microscopy (SEM) analysis, the biofilm culture was grown on the pegs of a transferable solid phase (TSP) lid (Nunc Immuno TSP, 445497) as described by Ceri et al.17 Antibiotic susceptibility of planktonic culture Antibiotic susceptibility tests for determining MICs and MBCs were performed using the microbroth dilution method according to CLSI guidelines.20 The MBC of the higher inoculum dose (Hi-MBC) of planktonic cells that corresponds to the cell density of matured biofilm was determined in the same manner except for the use of an inoculum size of 2 × 109 cfu/mL. The final result was the concordant value of the triplicate data set. Antibiotic susceptibility of V. cholerae O139 biofilm V. cholerae O139 biofilm formed in 96-well plates at different timepoints was treated with respective antibiotics in the range of 0.0156–2048 mg/L for 24 h at 37°C. After washing three times with PBS, each biofilm was mechanically disrupted, suspended in LB medium and plated on a plain LB agar plate for cell count determination. The lowest antibiotic concentration at which no viable cell count was obtained was recorded as the minimum biofilm eradication concentration (MBEC) for the respective antibiotics.17 For quantifying the biofilm resistance factor (BRF), the antibiotic concentration one doubling dilution below the MBEC was used, along with the formula proposed by Stewart.21 All assays were performed in three independent experiments. SEM analysis The lethal effect of antibiotic treatment on planktonic as well as biofilm cultures were analysed by SEM analysis. Briefly, the antibiotic-treated planktonic and biofilm cultures at their sub-MBC (ampicillin, 32 mg/L; ceftriaxone, 0.5 mg/L; ciprofloxacin, 0.0078 mg/L; doxycycline, 1 mg/L; erythromycin, 4 mg/L) and sub-MBEC (ampicillin, 2048 mg/L; ceftriaxone, 1024 mg/L; ciprofloxacin, 0.5 mg/L; doxycycline, 128 mg/L; erythromycin, 1024 mg/L) were fixed with 2.5% glutaraldehyde for 10 min and 1 h at room temperature, respectively. After washing twice with double-distilled water, the planktonic cultures were deposited on a 0.45 μm nitrocellulose membrane filter and subjected to drying. The pegs having biofilm were then detached from the lid and kept drying for 2–3 days. Dried samples were mounted on brass stubs and coated with gold in a Jeol JFC-1100 sputter coating unit. Images were acquired using a Quanta 400 environmental scanning electron microscope (ESEM/EDX). Analysis of antibiotic degradation and penetration Antibiotic degradation by biofilm culture was determined purely by a qualitative method, as described by Anderl et al.19 Briefly, the membrane-grown biofilms were placed on LB plates containing the MBEC of respective antibiotics and were incubated at 37°C overnight. After incubation, biofilms were removed and the plates were spread with 100 μL of planktonic V. cholerae culture (∼107 cfu/mL). The plates were observed for growth at the biofilm-exposed area. LB plates without antibiotic were used as a control. Antibiotic degradation by planktonic culture was determined by inoculating cultures grown overnight (∼109 cfu/mL) at 37°C with respective antibiotics at their 10× MIC. A 20 μL portion of the spent medium, collected every 30 min for 4 h, was added to the concentration disc (Hi-Media, sterile susceptibility testing disc, SD067) and then used for disc diffusion testing against V. cholerae O139. A calibration curve established with 2-fold dilutions, starting from 10× MIC of the respective antibiotics, and their corresponding median zone of inhibition was used for determining the relative antibiotic concentration in test discs. Antibiotic degradation rate was determined by plotting the natural log of the antibiotic concentration with respect to time. The antibiotic penetration through biofilm was performed as described by Anderl et al.19 Briefly, the membrane-grown biofilm, sandwiched between sterile membrane and moistened concentration disc, was placed on an LB plate containing the MBEC of the respective antibiotics and incubated for 24 h at 37°C. The control assemblies without biofilm were run in parallel. Thereafter, the disc was placed on an LB plate seeded with V. cholerae culture (∼107 cfu/mL) and incubated at 37°C for 24 h. The median zone of inhibition on an LB plate with test and control assemblies was determined and the antibiotic penetration through biofilm was expressed in terms of percentage, in relation to the values from control assemblies. The MIC values of respective antibiotics were directly used for antibiotic penetration into planktonic culture. Analysis of antibiotic-surviving population (ASP) from the biofilm The percentage of ASP was determined as the fraction of viable counts obtained from the antibiotic-treated biofilm at the sub-MBECs, as mentioned above, to the untreated control. These ASPs were subcultured for three generations and then their planktonic form again tested for their ability to grow in the presence of increased antibiotic concentrations (equal to MBC and MBEC). Two randomly picked clones from respective ASPs were again allowed to form biofilm and subjected to MBEC determination. Determination of spontaneous mutant frequency The occurrence of spontaneous mutants among planktonic, control biofilm and antibiotic-treated biofilm cultures was determined using a rifampicin-resistant phenotype as a reporter.22 The V. cholerae cells obtained from planktonic culture, antibiotic-treated (at sub-MBEC) biofilms and control biofilm, as described earlier, were plated on LB plates with and without rifampicin (100 mg/L) at appropriate dilutions. The plates were incubated at 37°C for 48 h. Spontaneous mutant frequency was calculated as the fraction of viable counts on the rifampicin plate relative to the viable counts on the LB plate without antibiotic. Detection of conditionally viable environmental cell (CVEC) phenotype The occurrence of CVEC in 48 h old biofilm cultures was determined with streptomycin (50 mg/L) selection.9 The biofilm culture was washed, homogenized in sterile PBS, plated onto LB plates with and without streptomycin and further incubated at 37°C for 24 h. The percentage reduction in the viable counts obtained on the streptomycin plate compared with the plain LB plate was calculated for the occurrence of the CVEC phenotype. Animal studies The infectivity status of ASP clones obtained from antibiotic-treated biofilm cultures was evaluated in the infant mouse model.23 Briefly, planktonic cultures of five random ASP clones (each one from respective antibiotic-treated biofilm cultures) along with WT were administered orally (2.5 × 105 cfu/mouse) to 4–5-day-old, infant BALB/c mice in a group of four. At 20–24 h post-infection the mice were sacrificed and the small intestine from each was removed and homogenized. The contents were then appropriately diluted in sterile PBS and plated onto thiosulphate–citrate–bile salts–sucrose agar (TCBS) plates for viable counts. Statistical analysis The experimental data were analysed using one-way ANOVA analysis for statistical significance. R2 (square of the Pearson’s product moment correlation coefficient) was determined using Microsoft Excel 2007. A P value of <0.05 was considered statistically significant for all the data analysis. For MIC, MBC and MBEC determination, means of concordant values from three independent experiments were taken as the results. Ethics All the animal infection studies were performed in accordance with the Institutional Animal Ethics Committee (IAEC) of the Defence Research and Development Establishment (Registration number 37/Go/C/1999/CPCSEA) and the animals were provided with food and water ad libitum according to the guidelines. Results Antibiotic effect on V. cholerae planktonic culture Antibiotics from five classes, namely ampicillin, doxycycline, ciprofloxacin, erythromycin and ceftriaxone (belonging to β-lactams, tetracyclines, fluoroquinolones, macrolides and cephalosporins), were chosen for the study. The effect of these antibiotics on V. cholerae O139 planktonic culture was measured in terms of MIC and MBC, as per CLSI guidelines,20 and values are summarized in Table 1. The effectiveness of azithromycin, the currently recommended antibiotic from the macrolide class, is presented in Table S1 (available as Supplementary data at JAC Online). Based on the MIC values, it could be inferred that the planktonic form of V. cholerae is readily susceptible to the tested antibiotics. The Hi-MBC was examined to understand the effect of inoculum size on antibiotic susceptibility. With the higher inoculum size, the MIC values could not be clearly determined using the microbroth dilution method, but the MBC values obtained were used for the comparison. The obtained Hi-MBC values (Table 1) were in the range of 2-fold higher as compared with standard inoculum size. Table 1. Antibiotic effect on planktonic and biofilm cultures Test method Ampicillin (mg/L) Ceftriaxone (mg/L) Ciprofloxacin (mg/L) Doxycycline (mg/L) Erythromycin (mg/L) CLSI MIC 16 0.125 0.015625 0.25 0.5 CLSI MBC 64 1 0.015625 2 8 Hi-MBC 128 1 0.03125 4 16 MBEC 8 h 1024 1 0.25 16 256 MBEC 12 h >2048 64 0.5 16 512 MBEC 24 h >2048 512 0.5 32 1024 MBEC 36 h >2048 >1024 0.5 128 1024 MBEC 48 h >2048 >1024 2 256 2048 Test method Ampicillin (mg/L) Ceftriaxone (mg/L) Ciprofloxacin (mg/L) Doxycycline (mg/L) Erythromycin (mg/L) CLSI MIC 16 0.125 0.015625 0.25 0.5 CLSI MBC 64 1 0.015625 2 8 Hi-MBC 128 1 0.03125 4 16 MBEC 8 h 1024 1 0.25 16 256 MBEC 12 h >2048 64 0.5 16 512 MBEC 24 h >2048 512 0.5 32 1024 MBEC 36 h >2048 >1024 0.5 128 1024 MBEC 48 h >2048 >1024 2 256 2048 CLSI MIC, MIC at 5 × 105 cfu/mL of planktonic culture; CLSI MBC, MBC at 5 × 105 cfu/mL of planktonic culture; Hi-MBC, MBC at 2 × 109 cfu/mL of planktonic culture; MBEC 8–48 h, MBEC for 8, 12, 24, 36 and 48 h old biofilm culture. Table 1. Antibiotic effect on planktonic and biofilm cultures Test method Ampicillin (mg/L) Ceftriaxone (mg/L) Ciprofloxacin (mg/L) Doxycycline (mg/L) Erythromycin (mg/L) CLSI MIC 16 0.125 0.015625 0.25 0.5 CLSI MBC 64 1 0.015625 2 8 Hi-MBC 128 1 0.03125 4 16 MBEC 8 h 1024 1 0.25 16 256 MBEC 12 h >2048 64 0.5 16 512 MBEC 24 h >2048 512 0.5 32 1024 MBEC 36 h >2048 >1024 0.5 128 1024 MBEC 48 h >2048 >1024 2 256 2048 Test method Ampicillin (mg/L) Ceftriaxone (mg/L) Ciprofloxacin (mg/L) Doxycycline (mg/L) Erythromycin (mg/L) CLSI MIC 16 0.125 0.015625 0.25 0.5 CLSI MBC 64 1 0.015625 2 8 Hi-MBC 128 1 0.03125 4 16 MBEC 8 h 1024 1 0.25 16 256 MBEC 12 h >2048 64 0.5 16 512 MBEC 24 h >2048 512 0.5 32 1024 MBEC 36 h >2048 >1024 0.5 128 1024 MBEC 48 h >2048 >1024 2 256 2048 CLSI MIC, MIC at 5 × 105 cfu/mL of planktonic culture; CLSI MBC, MBC at 5 × 105 cfu/mL of planktonic culture; Hi-MBC, MBC at 2 × 109 cfu/mL of planktonic culture; MBEC 8–48 h, MBEC for 8, 12, 24, 36 and 48 h old biofilm culture. V. cholerae biofilm and its antibiotic resistance V. cholerae O139 forms a robust biofilm on the microtitre plate format. The noticeable biofilm formation occurred as early as 8 h and matured by 48 h of incubation; after that, no significant increase in biofilm mass was observed (Figures S1 and S2). The biofilm formation and its corresponding cell density was found to be increasing up to 48 h and became constant at 72 h of incubation (Figure S1). The effect of antibiotics at different ages of V. cholerae O139 biofilm cultures, measured in terms of MBEC and BRF for all the five antibiotics tested, is summarized in Tables 1 and 2. Notably, for all the tested antibiotics, the MBEC of biofilm culture was significantly (P < 0.05) higher than the MBC of planktonic culture (Figure S3) and the BRF of the matured biofilm against the above antibiotics ranged from 70 (for ampicillin and ciprofloxacin) to 1300 (for ceftriaxone) (Table 2). With ampicillin and ceftriaxone, complete eradication of biofilm (>99.999% reduction) was not observed at the concentrations tested. Further, in all the cases, aged biofilm exhibited a higher antimicrobial resistance than younger biofilm. The biofilm of V. cholerae O1 El Tor biotype was also examined for its susceptibility and the results are summarized in Table S2. Though there was a minor variation in its biofilm formation kinetics, as compared with the V. cholerae O139 strain (Figure S4), the biofilm of this strain also exhibited increased antibiotic resistance relative to its planktonic counterpart. The increase in antibiotic resistance from the planktonic to the biofilm form may have clinical significance in vivo by affecting the treatment outcome; e.g. treatment failures with ciprofloxacin were associated with V. cholerae strains showing increased in vitro MICs even within the susceptible breakpoint range. Table 2. BRF, ASP, penetration and degradation parameters of individual antibiotics Parameter Ampicillin Ceftriaxone Ciprofloxacin Doxycycline Erythromycin BRF 70 1300 70 80 100 ASPB 0.95 0.037 0.012 0.18 0.018 PenetrationB 92.8 95 100 84.6 93.75 DegradationB none occurred none occurred none occurred none occurred none occurred Degradationp (μg/h) 0.063 0.040 0.021 0.11 0.344 Parameter Ampicillin Ceftriaxone Ciprofloxacin Doxycycline Erythromycin BRF 70 1300 70 80 100 ASPB 0.95 0.037 0.012 0.18 0.018 PenetrationB 92.8 95 100 84.6 93.75 DegradationB none occurred none occurred none occurred none occurred none occurred Degradationp (μg/h) 0.063 0.040 0.021 0.11 0.344 ASPB, percentage of ASP from biofilm culture treated at sub-MBEC of respective antibiotics (ampicillin, 2048 mg/L; ceftriaxone, 1024 mg/L; ciprofloxacin, 0.5 mg/L; doxycycline, 128 mg/L; erythromycin, 1024 mg/L); penetrationB, percentage of antibiotic penetrated through biofilm culture in comparison with control in biofilm penetration assay; degradationB, zone of growth occurred due to the degradation of antibiotic (at MBEC) by biofilm culture; degradationp, degradation of antibiotics by planktonic culture (μg/h), which was not significant (P > 0.05) compared with control MH broth without planktonic culture. Table 2. BRF, ASP, penetration and degradation parameters of individual antibiotics Parameter Ampicillin Ceftriaxone Ciprofloxacin Doxycycline Erythromycin BRF 70 1300 70 80 100 ASPB 0.95 0.037 0.012 0.18 0.018 PenetrationB 92.8 95 100 84.6 93.75 DegradationB none occurred none occurred none occurred none occurred none occurred Degradationp (μg/h) 0.063 0.040 0.021 0.11 0.344 Parameter Ampicillin Ceftriaxone Ciprofloxacin Doxycycline Erythromycin BRF 70 1300 70 80 100 ASPB 0.95 0.037 0.012 0.18 0.018 PenetrationB 92.8 95 100 84.6 93.75 DegradationB none occurred none occurred none occurred none occurred none occurred Degradationp (μg/h) 0.063 0.040 0.021 0.11 0.344 ASPB, percentage of ASP from biofilm culture treated at sub-MBEC of respective antibiotics (ampicillin, 2048 mg/L; ceftriaxone, 1024 mg/L; ciprofloxacin, 0.5 mg/L; doxycycline, 128 mg/L; erythromycin, 1024 mg/L); penetrationB, percentage of antibiotic penetrated through biofilm culture in comparison with control in biofilm penetration assay; degradationB, zone of growth occurred due to the degradation of antibiotic (at MBEC) by biofilm culture; degradationp, degradation of antibiotics by planktonic culture (μg/h), which was not significant (P > 0.05) compared with control MH broth without planktonic culture. Increased biofilm antibiotic resistance was not correlated with antibiotic parameters The inherent properties of antibiotics such as molecular size, stability and penetrability were not found to be correlated with the increased antibiotic resistance exhibited by the biofilm. The BRF (Table 2) and the molecular size of the antibiotics were poorly correlated (R2 = 0.06) (Figure S5). Further, from the antibiotic degradation assay, it was found that the stability of the antibiotics was unchanged in the presence of planktonic as well as biofilm cultures of V. cholerae O139 (Table 2). The ability of antibiotics to penetrate into the planktonic cell was found to be in the following order: ciprofloxacin > ceftriaxone > doxycycline > erythromycin > ampicillin, as inferred from their MIC values, whereas the order was slightly changed for biofilm penetration and found to be as follows: ciprofloxacin > ceftriaxone > erythromycin > ampicillin > doxycycline (Table 2). However, it could not be interrelated with the increase in antimicrobial resistance exhibited by the biofilm of V. cholerae O139 (R2 = 0.0365). Biofilm safeguards against antibiotic stress Bacterial cultures exposed to antibiotic stress undergo various morphological changes before growth stalls and/or death occurs.24 Exposure to antibiotics in planktonic culture resulted in various morphological changes such as spheroplast formation (ampicillin and ceftriaxone), elongation or filamentation (ciprofloxacin), and small bulging and shortening of cells (doxycycline and erythromycin) (Figure 1a and Figure S6a). However, the biofilm counterpart was found to be unaltered in morphology (Figure 1b and Figure S6b). Therefore, it could be inferred that the planktonic culture was more vulnerable to antibiotic stressors than biofilm culture. Figure 1. View largeDownload slide SEM analysis of antibiotic-treated planktonic and biofilm cultures of V. cholerae O139. (a) Planktonic cells were treated with respective antibiotics at their sub-lethal concentration and deposited on 0.45 µm nitrocellulose membrane after fixing with glutaraldehyde (2.5%) and images were acquired at ×5000 (insert image: ×20 000). (b) Biofilm cultures grown on the pegs of a transferable solid phase (TSP) lid were treated with respective antibiotics at their sub-lethal concentration, fixed in 2.5% glutaraldehyde and images were acquired at ×10 000. Figure 1. View largeDownload slide SEM analysis of antibiotic-treated planktonic and biofilm cultures of V. cholerae O139. (a) Planktonic cells were treated with respective antibiotics at their sub-lethal concentration and deposited on 0.45 µm nitrocellulose membrane after fixing with glutaraldehyde (2.5%) and images were acquired at ×5000 (insert image: ×20 000). (b) Biofilm cultures grown on the pegs of a transferable solid phase (TSP) lid were treated with respective antibiotics at their sub-lethal concentration, fixed in 2.5% glutaraldehyde and images were acquired at ×10 000. Increased biofilm resistance is a transient phenotype in V. cholerae The primary question raised regarding increased biofilm resistance was whether it is an inheritable one due to the accumulation of genetic changes or just a transient phenotype associated with biofilm formation. If any inheritable changes that lead to the selection of antibiotic resistance clones occurred among the ASP, they should also be expressed in subsequent generations. Therefore, ASPs that occurred at a rate of 0.012%–0.95% (Table 2) among the biofilm cultures challenged at sub-MBEC of respective antibiotics were analysed. The ASP clones tested, consisting of 20 random clones from each antibiotic treatment, were found to be susceptible to the antibiotics at their respective MBC and MBEC in planktonic culture. Further, when they were allowed to form biofilm and then subjected to MBEC assay, they exhibited similar resistance profiles to that of the parent WT. Spontaneous mutants that confer resistance to rifampicin occurred at a frequency of 2.31 × 10−9 among biofilm culture that was ∼12.3 times higher than that of its planktonic culture (Table 3). However, the rifampicin mutant frequency among ASP clones was found to be zero. This may rule out the possibility of hypermutation among ASP clones, which may be considered a reason for increased antibiotic resistance. Additionally, we could not find any CVEC-like phenotype among 48 h old biofilm culture through the streptomycin selection assay (data not shown). Table 3. Frequency of spontaneous mutants that confer resistance to rifampicin among planktonic, biofilm and antibiotic-challenged biofilm cultures Culture type Rifampicin mutant frequencya Planktonic culture 2.31 ± 1.6 × 10−9 Biofilm culture 2.84 ± 2.0 ×10−8 Biofilmb+ampicillin (2048 mg/L) 0 Biofilmb+ceftriaxone (1024 mg/L) 0 Biofilmb+ciprofloxacin (0.5 mg/L) 0 Biofilmb+doxycycline (128 mg/L) 0 Biofilmb+erythromycin (1024 mg/L) 0 Culture type Rifampicin mutant frequencya Planktonic culture 2.31 ± 1.6 × 10−9 Biofilm culture 2.84 ± 2.0 ×10−8 Biofilmb+ampicillin (2048 mg/L) 0 Biofilmb+ceftriaxone (1024 mg/L) 0 Biofilmb+ciprofloxacin (0.5 mg/L) 0 Biofilmb+doxycycline (128 mg/L) 0 Biofilmb+erythromycin (1024 mg/L) 0 a Spontaneous mutants (that confer resistance to rifampicin) were selected on LB plates containing rifampicin (100 mg/L). b Biofilm cultures treated with respective antibiotics at their sub-MBEC concentration were selected on LB plates containing rifampicin (100 mg/L). Table 3. Frequency of spontaneous mutants that confer resistance to rifampicin among planktonic, biofilm and antibiotic-challenged biofilm cultures Culture type Rifampicin mutant frequencya Planktonic culture 2.31 ± 1.6 × 10−9 Biofilm culture 2.84 ± 2.0 ×10−8 Biofilmb+ampicillin (2048 mg/L) 0 Biofilmb+ceftriaxone (1024 mg/L) 0 Biofilmb+ciprofloxacin (0.5 mg/L) 0 Biofilmb+doxycycline (128 mg/L) 0 Biofilmb+erythromycin (1024 mg/L) 0 Culture type Rifampicin mutant frequencya Planktonic culture 2.31 ± 1.6 × 10−9 Biofilm culture 2.84 ± 2.0 ×10−8 Biofilmb+ampicillin (2048 mg/L) 0 Biofilmb+ceftriaxone (1024 mg/L) 0 Biofilmb+ciprofloxacin (0.5 mg/L) 0 Biofilmb+doxycycline (128 mg/L) 0 Biofilmb+erythromycin (1024 mg/L) 0 a Spontaneous mutants (that confer resistance to rifampicin) were selected on LB plates containing rifampicin (100 mg/L). b Biofilm cultures treated with respective antibiotics at their sub-MBEC concentration were selected on LB plates containing rifampicin (100 mg/L). ASP clones were not affected in their infectivity An ASP clone from each antibiotic-treated biofilm culture was evaluated along with their parent WT for their ability to infect mouse intestine. They were able to produce cholera-like symptoms upon infection and were similar in their ability to colonize mouse intestine compared with their parent WT strain; there was no statistical difference among them in their mean log10 cfu/small intestine (P > 0.05) (Figure 2). Figure 2. View largeDownload slide Infant mouse intestinal colonization with WT and ASP clones. The colonization ability (mean ± SD log10 cfu/small intestine) of ASP clones was not statistically different compared with WT (P > 0.05). ∗ASP clones were obtained from respective antibiotic-challenged biofilm culture as described in the Materials and methods section. AMP, ampicillin; CIP, ciprofloxacin; CRO, ceftriaxone; DOX, doxycycline; ERY, erythromycin. Figure 2. View largeDownload slide Infant mouse intestinal colonization with WT and ASP clones. The colonization ability (mean ± SD log10 cfu/small intestine) of ASP clones was not statistically different compared with WT (P > 0.05). ∗ASP clones were obtained from respective antibiotic-challenged biofilm culture as described in the Materials and methods section. AMP, ampicillin; CIP, ciprofloxacin; CRO, ceftriaxone; DOX, doxycycline; ERY, erythromycin. Discussion The cholera pathogen V. cholerae O139 forms a robust biofilm and it is very evident from our study that the antibiotic resistance exhibited by the biofilm culture of this bacterium is several-fold higher than its planktonic counterpart (P < 0.05), regardless of the antibiotic type tested. The term ‘resistance’ used here is to make a distinction between the susceptible planktonic counterpart; it is not referring to the conventional resistance that is linked to a specific genetic determinant. This kind of increased resistance was also found with the biofilm of V. cholerae O1 El Tor biotype (Table S2), which implies that this unique phenomenon might be universal for all biofilm-forming V. cholerae strains. This unconventional biofilm antibiotic resistance was not found to be associated with either the increased cell number or the physical intactness of biofilm structure. Further, the antibiotic characteristics such as molecular size, stability and penetrating ability through the biofilm were also found to be insignificant for the increased resistance. The presence of the persister phenotype within the V. cholerae biofilm could be one of the reasons associated with increased antibiotic resistance as the same phenomenon was observed in other biofilm-forming bacteria also.14 The antibiotic resistance exhibited by the biofilm culture appeared to be exclusive to the biofilm phenotype because when the ASP clones obtained from the antibiotic-treated biofilm were tested in their planktonic as well as their biofilm form they exhibited increased resistance only in their biofilm culture and resumed their susceptibility status in their planktonic form. Further, these ASP clones were found to be similar to their parent WT in their ability to colonize mouse intestine (Figure 2). This finding supports the view regarding antibiotic escape and infectivity due to biofilm formation, which may be seen as the equivalent version of relapsing in other chronic infections.25 Concerning the protective role conferred by the biofilm of V. cholerae, it was observed that the antibiotic-induced stress exerted on planktonic cells was found to be nullified in the biofilm niche and the cells were found to be free from morphological changes such as spheroplast formation, filamentation, cell elongation, shortening and bulging (Figure 1). The occurrence of heterogeneous phenotypes among the biofilm residents could be a reason for their buffering capacity. The heterogeneity among the biofilm cells was also shown to be responsible for the emergence of persister cells that were unaffected by the antimicrobial actions.14,25,26 The ASP from the respective antibiotic-treated biofilm may explain the phenomenon of persister-associated resistance in the V. cholerae biofilm; however, further studies are required to conclude that this is the only mechanism responsible for this increased antibiotic resistance. The occurrence of spontaneous mutants in the untreated V. cholerae biofilm culture was found to be > 12-fold higher than the planktonic culture. However, the absence of spontaneous mutants (that confer resistance to rifampicin) among the antibiotic-treated biofilm culture suggest that whatever resistance exhibited by the biofilm may not have resulted from spontaneous mutation. It has been observed with Pseudomonas aeruginosa and other bacteria that the rate of spontaneous mutation for antibiotics decreases with the increasing concentration of antibiotics tested;27,28 this could be the reason for the lack of rifampicin mutants among the ASP clones as the biofilm cultures were exposed to very high concentration of antibiotics. We also reasoned that the occurrence of the CVEC phenotype could be a possible mechanism for the increased biofilm resistance; however, the absence of this particular phenotype in the 48 h old biofilm culture excluded this possibility. Although the above findings were in favour of linking the biofilm phenotype to the increased antibiotic resistance, the involvement of genotypic changes could not be conclusively ruled out, as it requires more genetic analysis before arriving at this conclusion. The conventional antibiotic resistance among MDR V. cholerae strains has already been reported for all the front-line drugs including ciprofloxacin and azithromycin.29,30 In this scenario, the unconventional biofilm resistance, as observed in this study to all the tested antibiotics including ciprofloxacin and azithromycin, poses further challenges regarding their usage. Although the effectiveness of antimicrobial treatments in controlling biofilm discharge through the stools of cholera patients needs a detailed understanding, the possibility of biofilm-associated antimicrobial escape cannot be ruled out. Therefore, wherever biofilm is known to be involved, a standard planktonic MIC-based treatment regimen may not be effective and consideration of an MBEC-based dose regimen, wherever possible, may be more useful; however, further studies are warranted before implementing this approach in the clinical set-up. Acknowledgements We are thankful to the Director of the Defence Research and Development Establishment, Gwalior, India for providing all the facilities and financial support to carry out this work. Funding This study was supported by institutional funds provided by the Defence Research and Development Establishment, Gwalior, India. Transparency declarations None to declare. Supplementary data Tables S1 and S2 and Figures S1 to S6 are available as Supplementary data at JAC Online. References 1 Ali M , Nelson AR , Lopez AL et al. Updated global burden of cholera in endemic countries . PLoS Negl Trop Dis 2015 ; 9 : e0003832. Google Scholar CrossRef Search ADS PubMed 2 Black RE. The prophylaxis and therapy of secretory diarrhea . Med Clin North Am 1982 ; 66 : 611 – 21 . Google Scholar CrossRef Search ADS PubMed 3 Lindenbaum J , Greenough WB , Islam MR. Antibiotic therapy of cholera . Bull World Health Organ 1967 ; 36 : 871 – 83 . 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