phenotypic investigation of paired pseudomonas

Доклади на Българската академия на науките Comptes rendus de l’Acad´ emie bulgare des Sciences Tome 71, No 8, 2018

BIOLOGY Microbiology

PHENOTYPIC INVESTIGATION OF PAIRED PSEUDOMONAS AERUGINOSA STRAINS ISOLATED FROM CYSTIC FIBROSIS PATIENTS PRIOR- AND POST-TOBRAMYCIN TREATMENT Dayana Borisova, Tanya Strateva∗ , Tsvetelina Paunova-Krasteva, Ivan Mitov∗ , Stoyanka Stoitsova (Submitted on May 29, 2017)

Abstract Cystic fibrosis is a hereditary disease accompanied by extensive secretion of viscous mucins in the digestive and respiratory tracts. Bacterial infections, very often caused by Pseudomonas aeruginosa, are the leading cause for exacerbations and early death of patients. One of the comparatively effective to date antibiotics against P. aeruginosa is tobramycin. In spite of the initial success of the treatment, however, some part of the bacterial population remains persistent in the lungs. The comparison of paired strains isolated from the same patient prior- and post-antibiotic treatment would elucidate some adaptive mechanisms related with the bacterial persistence in cystic fibrosis, which determined the objectives of the present study. We performed a comparative phenotypic investigation on a panel of 6 couples of paired strains, each couple containing a strain isolated prior- and a strain isolated post-tobramycin treatment from the same patient. We characterized the strains’ growth parameters, biofilm formation, and motility (swimming, swarming, and twitching). Strain-to-strain differences were registered with most of the tests. As a distinct trend demonstrated in the examined pairs, the changes in growth parameters should be underlined, and especially – the prolongation of the lag phase of the post-treatment strains. This could be an adaptation for persistence. Key words: Pseudomonas aeruginosa paired strains, tobramycin, phenotypic adaptations This study was supported by “Programme for Carrier Development of Young Scientists”, BAS, Contract No DFNP-55/27.04.2016. DOI:10.7546/CRABS.2018.08.05

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Introduction. Cystic fibrosis (CF) is a genetic disorder, a consequence of the deficiency of the CF transmembrane conductance regulator. The loss of this protein’s function causes alterations in many organs, among them the most serious health problems are associated with the lungs [1 ]. The accumulated sputum in the CF lungs is a good environment for microbial colonization [2 ]. Pseudomonas aeruginosa is the most prevalent pathogen among CF patients. One of the comparatively effective to date antibiotics against P. aeruginosa is tobramycin. In spite of the initial success of the treatment, however, some part of the bacterial population remains persistent in the airways. The long-term persistence of this bacterium in the specific environment of the CF lung is associated with phenotypic and genotypic diversification which is considered to generate phenotypes specifically adapted to the CF airway condition [2, 3 ]. Most of the current knowledge on the phenotypic diversification and the adaptations of P . aeruginosa to chronic persistence in the CF lung arises from comparisons of longitudinal isolates – strains from the same patient collected at various stages of the disease [4 ]. While it is suggested that one of the factors resulting in bacterial adaptation is the regular exposure to different antibiotics [3 ], this statement has not found much experimental support. This determined the aim of the present study: to perform a phenotypic comparison between pairs of clinical CF strains of P. aeruginosa collected from the same patient prior- and post-inhalatory treatment with tobramycin. We addressed growth parameters and biofilm formation in three cultivation media as well as the motility of the strains. Materials and methods. Strains and cultivation. Six pairs of P. aeruginosa clinical strains were included in the study. Each pair originated from the same patient and contains a strain isolated prior- and a strain isolated postinhalatory treatment with tobramycin (Table 1). The strains were maintained as stocks in 30% glycerol at −80 ◦ C. For the phenotypic experiments, the bacteria were cultivated in one of the following media: LB broth (10 g/L of Bacto triptone (Difco), 5 g/L of yeast extract (Difco) and 10 g/L of NaCl); Mueller–Hinton broth (MH) (BulBio-NCIPD); M63 minimal salt medium (0.02 M KH2 PO4 , 0.04 M K2 HPO4 , 0.02 (NH4 )2 SO4 , 0.1 mM MgSO4 , 0.04 M glucose). Table

1

Strain couples included in the study Patient No

C. R. Acad. Bulg. Sci., 71, No 8, 2018

PaT-6

PaT-7

PaT-8

PaT-9

PaT-10

PaT-11

PaT-12

6

PaT-5

5

PaT-4

4

PaT-3

3

PaT-2

Number of tobramycin inhalatory cycles prior to the isolation of the strain

2

PaT-1

Strain number

1

0

3

0

3

0

15

0

2

0

2

0

3

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Estimation of bacterial growth. Overnight cultures of the strains were diluted 1:100 in the respective test medium. 150 µl of bacterial suspension were applied into the wells of 96-well U-shaped plates. Each variant was repeated in 6 wells. At intervals, the plates were shaken shortly and absorbance was measured on plate reader at 620 nm. Biofilm growth. It was evaluated by the crystal violet assay [5 ]. Briefly, plates loaded as above were cultivated for 24 h, the unattached bacteria were withdrawn, and the wells were washed with PBS. Then the wells were coloured for 15 min with 0.1% crystal violet, washed extensively and solubilised in 70% ethanol. The absorbance was measured at 570 nm wavelength. Motility assays. Motility plates were prepared using tryptic soy broth (TSB) (Difco) solidified by different amounts of agar. Swimming motility was estimated on 0.3% agar. Swarming motility was tested in 0.6% agar. Twitching motility was tested on 1% agar. For all tests, the bacteria were inoculated with sterile toothpick in the middle of the plate, incubation was performed for 24 h at 37 ◦ C. The diameters of the swimming and swarming motility zones were measured. For twitching motility visualisation, the agar was removed from the plates, then 0.1% crystal violet was applied for 15 min, and the excess of the dye was washed. Results and discussion. The examination of growth parameters was performed with time, within 48 hours. Growth curves were prepared and analysed with regard to the maximum culture density (ODmax measured at 620 nm wavelength), and the length of the lag phase. Following the changes of the OD, we compared the growth parameters of the paired isolates in three growth media: LB (a medium very often used in the studies of P. aeruginosa), MH (medium recommended for the study of antibiotic sensitivity) and M63 minimal salt medium supplemented with glucose as a carbon source. When the ODmax of the strains acquired upon reaching the stationary phase is considered, there was a significant strain-to-strain variability, and we observed strain specific preferences to the growth medium. In only one strain couple, PaT-11 and PaT-12, we could register a strict trend – lower values of ODmax in all three media for the post-treatment isolate (Fig. 1). Such a strain-to-strain variability of ODmax is in accordance with a previous observation on a large P. aeruginosa reference CF strain panel [4 ]. Of importance, with most of the strain couples, and in all media studied, a well-expressed trend for prolongation of the lag-phase was determined (Table 2). The lag phase is the period of adaptation of the bacterial cells to a changed environment [6, 7 ]. While no increase in bacterial number is registered [6 ], it has been shown that the bacteria in the lag-phase are metabolically active [7, 8 ] and undergo processes preparing the culture to enter into the exponential phase of growth. Upon a previous examination of CF longitudinal isolates of P. aeruginosa, it has been noted that the strains considered to be best adapted to chronic infection are characterized by a very long lag-phase [4 ]. The present observation 1046

D. Borisova, T. Strateva, Ts. Paunova-Krasteva et al.

Fig. 1. Growth of strain couple PaT-11 and PaT-12 in LB broth /LB/ (A), Mueller-Hinton broth /MH/ (B) and M63 minimal medium /M63/ (C)

for prolongation of the lag-phase of post-tobramycin treatment strains indicates an impact of the antibiotic action on this growth parameter. It is quite probable that this result is related to the phenomenon of persistence. It is accepted that persisters are not mutants but phenotypic variants of regular bacterial cells [9 ] that survive the antibiotic action due to their state of lower metabolic activity or dormancy [10–12 ]. Once the antibiotic is no longer available, the persister cells may produce both metabolically active and inactive cells [14 ]. The present results imply that the here examined post-treatment strains may have originated from persister cells, the prolonged lag-phase could be both a consequence of the antibiotic action, and an adaptation for survival of the bacterial populations under unfavourable conditions. One important adaptive mechanism of P. aeruginosa during colonization of the CF lung is the formation of biofilms. This process is as well considered one of the factors in the bacterial persistence in the infected lung [2 ]. To test the putative impact of the tobramycin treatment on this phenotype, we cultivated biofilms on 96-well plates in the three test media (LB, MH and M63) and applied the crystal violet assay [5 ]. The comparison between the pre- and post-tobramycin C. R. Acad. Bulg. Sci., 71, No 8, 2018

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Table

2

Duration of the lag-phase (hours). A general trend for prolongation of the lag-phase in the strains isolated post-treatment with tobramycin (shaded) is observed. Exception – strain couple PaT-5 and PaT-6

❳❳ ❳❳❳Medium ❳❳ Strain ❳❳

LB

MH

M63

PaT-1 PaT-2 PaT-3 PaT-4 PaT-5 PaT-6 PaT-7 PaT-8 PaT-9 PaT-10 PaT-11 PaT-12

5 5 3 5 5 3 3 6 5 6 4 7

5 6 4 6 7 3 4 6 5 6 3 7

6 7 6 7 7 4 7 7 7 7 6 7

treatment isolates showed no strict pattern of change. In two of the strain couples the post-treatment isolate tended to produce less biofilm (Fig. 2A, B). Two other strain couples showed the opposite tendency in two of the tested media (Fig. 2C, D). The remaining two pairs showed no strict trend and varied in the different media. Biofilm formation is a complicated, multi-factorial process. In a study of 43 CF and non-CF isolates, we have previouly shown that strains may express Table

3

Motility tests for swimming, swarming and twitching motility: dimensions of the motility zones Strain PaT-1 PaT-2 PaT-3 PaT-4 PaT-5 PaT-6 PaT-7 PaT-8 PaT-9 PaT10 PaT-11 PaT-12

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Motility Swimming 3.25 ± 0.01 3.34 ± 0.23 3.20 ± 0.12 3.23 ± 0.17 2.5 ± 0.01 3.57 ± 0.12 3.97 ± 0.07 2.48 ± 0.05 1.06 ± 0.012 1.31 ± 0.12 3.16 ± 0.28 2.12 ± 0.25

zone diameter (cm) Swarming Twitching 0.91 ± 0.16 – 0.82 ± 0.14 1.8 0.89 ± 0.07 1 0.96 ± 0.07 0.8 0.68 ± 0.06 – 1.02 ± 0.13 – 0.65 ± 0.1 – 0.80 ± 0.06 – 0.65 ± 0.016 – 0.62 ± 0.05 – 0.92 ± 0.16 – 0.78 ± 0.15 –


Fig. 2. Comparison of the biofilm growth by the strain couples in the three tested media. In two of the couples (A, F) a trend for biofilm diminution in the post-treatment isolates is observed; Two other couples (C, E) show a trend of biofilm increase in the post-treatment strain; (B, D) Growth medium-related differences

growth medium preferences, but may as well differ in the dynamics of biofilm growth and/or detachment, irrespective of the source of their isolation – CF or non-CF [4 ]. Summing up, the present results on biofilm formation show a strain specificity that is not strictly co-related with the effects of the tobramycin treatment. We also addressed three motility phenotypes. Two of them depend on the expression of flagella, i.e., swimming and swarming. Swimming motility is performed via flagella. So is swarming motility, however, it is associated with hyperflagelation [14 ]. We found out that regarding these two phenotypes, the preand post-treatment strains were alike (Table 3). As a rule, all the tested isolates were poor swarmers. Reduction of either swarming, or of both swarming and C. R. Acad. Bulg. Sci., 71, No 8, 2018

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swimming motility is considered to be an adaptive character of P. aeruginosa isolates from chronic infections [2 ], including these from CF [4 ]. This can also be considered a character of the here examined strain collection. Twitching motility is a result of the extension, tethering and retraction of type IV pili. We found evidence of twitching in only three of the strains (Table 3). This phenotype is more rarely found in CF strains of P. aeruginosa [4 ]. The gene pilB associated with the type IV pili production has been registered in 26.9% of the clinical isolates of P. aeruginosa, however, its incidence among CF isolates is considerably lower [15 ]. This is also in accordance with the present phenotypic data. Conclusions. The present study describes the results of a comparative phenotypic investigation on a panel of 6 couples of paired strains, each couple containing strains isolated from the same patient prior- and post-tobramycin treatment. We characterized the strains’ growth parameters and biofilm formation in three growth media, as well as motility (swimming, swarming, and twitching). The strain-to-strain differences registered with most of the tests showed no correlation with tobramycin treatment of the patients. As an exception, one distinct trend was demonstrated in the examined pairs: the prolongation of the lag phase of the post-treatment strains. Such a strict pattern of modulation of this growth parameter could be considered an adaptation for persistence.

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[8 ] Rolfe M., C. Rice, S. Lucchini, C. Pin, A. Thompson et al. (2012) Lag phase is a distinct growth phase that prepares bacteria for exponential growth and involves transient metal accumulation, J. Bacteriol., 194(3), 686–701. [9 ] Dorr T., M. Vulic, K. Lewis (2010) Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli, PLoS Biology, 8(2), e1000317. [10 ] Maissonneuve E., L. Shakespeare, M. Jorgensen, K. Gerdes (2011) Bacterial persistence by RNA endonucleases, PNAS, 108(32), 13206–13211. [11 ] Joers A., N. Kaldalu, T. Tenson (2010) The frequency of persisters in Escherichia coli reflects the kinetics of awakening from dormancy, J. Bacteriol., 192(13), 3379–3384. [12 ] Krylov V., O. Shaburoiva, E. Pleteneva, M. Bourkaltseva, S. Krylov et al. (2016) Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to children suffering with cystic fibrosis, Frontiers Microbiol., 7, Article 1631. [13 ] Rotem E., A. Loinger, I. Ronin, I. Levin-Reisman, C. Gabay et al. (2010) Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence, PNAS, 107(28), 12541–12546. [14 ] Kearns D. (2010) A field guide to bacterial swarming motility, Nat. Rev. Microbiol., 8, 634–644. [15 ] Mitov I., T. Strateva, B. Markova (2010) Prevalence of virulence genes among Bulgarian nosocomial and cystic fibrosis isolates of Pseudomonas aeruginosa, Brazilian J. Microbiol., 41, 588–595. The Stephan Angeloff Institute of Microbiology Bulgarian Academy of Sciences Acad. G. Bonchev St, Bl. 26 1113 Sofia, Bulgaria e-mail: [email protected]

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∗

Department of Medical Microbiology Faculty of Medicine Medical University of Sofia 1, St. Georgi Sofiiski Blvd 1431 Sofia, Bulgaria

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