Effect of freezing sputum on Pseudomonas aeruginosa population heterogeneity

JCF-01431; No of Pages 5

Journal of Cystic Fibrosis xx (2017) xxx – xxx www.elsevier.com/locate/jcf

Short Communication

Effect of freezing sputum on Pseudomonas aeruginosa population heterogeneity☆ Ali Poonja a , Alya Heirali a , Matthew Workentine b , Douglas G. Storey a,c , Ranjani Somayaji a,d , Harvey R. Rabin a , Michael G. Surette a,e , Michael D. Parkins a,d,⁎ a

Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Canada b Department of Veterinary Sciences, University of Calgary, Calgary, AB, Canada c Department of Biological Sciences, University of Calgary, Calgary, AB, Canada d Department of Medicine, University of Calgary, Calgary, AB, Canada e Department of Biochemistry, McMaster University, Hamilton, ON, Canada Received 26 September 2016; revised 14 December 2016; accepted 10 January 2017 Available online xxxx

Abstract Pseudomonas aeruginosa develops profound population heterogeneity in CF airways. How changes in these populations relate to clinical status is unknown. In order to facilitate this understanding, frequent sampling of this community is required. To determine if the collection and storage of sputum at home may pose a viable option, we collected sputum from ten patients. Sputum samples were partitioned in two, with half immediately processed on MacConkey agar and half assessed after freezing for one week in a home-freezer. From each sample, 88 isolates were assessed for antibiotic susceptibility and virulence factor production. Freezing resulted in a 103 CFU/ml drop in P. aeruginosa. However, across 1760 isolates, no consistent difference in either antibiotic susceptibility nor virulence factors was observed suggesting freezing induced indiscriminate killing. Home collection and freezing of sputum will enable frequent and convenient assessment of P. aeruginosa population dynamics in CF. © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Cystic fibrosis (CF) is characterized by chronic lower airways infection. The archetypal CF pathogen, Pseudomonas aeruginosa, eventually infects 60–80% of patients [1,2]. Infection with P. aeruginosa is associated with increased rates of lung function decline, exacerbation frequency and progression to end stage lung disease [3,4].

Q4

☆ The authors have no conflicts related to the above work. AP, AH, RS, and DGS have no disclosures to report. MDP, HRR, and MGS have received research support from Gilead Sciences. AP, AH performed the experiments herein. MW, MGS and MDP designed the study and secured funding. AH, RS, DGS and HRR assisted in the study design and data analysis. MDP serves as the guarantor of the work. This work was supported from a grant from Gilead Sciences. This work was presented in abstract form at the North American Cystic Fibrosis Conference in Orlando, FL, Oct 29, 2016. ⁎ Corresponding author at: Department of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada. E-mail address: [email protected] (M.D. Parkins).

Traditional models of research and care have extrapolated characteristics (including susceptibility testing) of individual isolates of P. aeruginosa derived from respiratory samples to chronically colonizing populations. It has since been observed, however, that chronically infecting populations are tremendously heterogeneous with respect to phenotypes including antibiotic susceptibility, virulence capacity and growth [5–9]. Clinical outcomes of anti-Pseudomonal therapies, not surprisingly, therefore, poorly correlate with antibiogram directed therapy against individual isolates [10,11]. How these diverse communities of P. aeruginosa persist and adapt to changing environmental influences, and how changes in the community affect the host are hereto unknown. While changes in total P. aeruginosa population density have not been shown to cause pulmonary exacerbations it is possible that a bloom in a select subpopulation may do so [12]. To understand if and how flux in P. aeruginosa populations affect

http://dx.doi.org/10.1016/j.jcf.2017.01.004 1569-1993© 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Poonja A, et al, Effect of freezing sputum on Pseudomonas aeruginosa population heterogeneity, J Cyst Fibros (2017), http://dx.doi.org/ 10.1016/j.jcf.2017.01.004

2

A. Poonja et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx

negative controls were used. High throughput techniques, developed by Workentine et al., were used to screen 1760 isolates for production of four virulence factors (proteases, siderophore, hemolysis and rhamnolipids) and susceptibility to antibiotics (aztreonam, meropenem, ciprofloxacin, and tobramycin) to understand effects of freezing on sub-population composition [6]. Isolates were grown in BHI broth for 24 h prior to performing virulence factor assays. Next, isolates were stamped onto plates containing media specific for the virulence mechanism being tested using a 96-pin replicator. Protease (PRO) and siderophone (CAS)-testing media were incubated for 18 h at 30 °C [6]; hemolysis testing was performed on Columbia Blood Agar (CBA) for 44 h at 37 °C [3]; rhamnolipid (RHA)-testing media for 48 h at 37 °C [15]. Photos were taken of CAS, CBA, and PRO plates using a Nikon D5100 camera and ControlMyNikon (Tetherscript Technology Canada, Vancouver) software. RHA plates were stored at 4 °C for an additional 24 h to help define halos and optimize downstream image analysis. Raw images were processed using Fiji Image J [16]. Each experiment was done in triplicate, and mean halo diameter value and SEM was recorded using a custom design plugin. For antibiotic susceptibility testing, isolates were grown in Muller Hinton (MH) broth for 24 h at 37 °C. Isolates were then stamped into media containing the following antibiotic concentrations: aztreonam (USP, Maryland) (ATM; 8, 32, 128 μg/ml), ciprofloxacin (Sandoz Canada, Quebec) (CIP; 4, 16, 64 μg/ml), meropenem (AstraZeneca, Ontario) (MEM; 4, 16, 64, μg/ml) and tobramycin (Novartis, Quebec) (TOB; 4, 16, 64, μg/ml) [6]. A Victor2 measured growth in each well with a positive confirmation being achieved for an OD ≥ 0.1 at 600 nm. Mean differences in virulence factor activity between the IC and FC were compared using paired t-tests and reported with 95% confidence intervals. Assessment of community antibiotic susceptibility was performed by comparing the mean proportion of susceptible isolates to the proportion of resistant isolates using CLSI established breakpoints for each antibiotic using paired t-tests [17]. All hypotheses were two-tailed at an α of 0.05, and we additionally addressed potential for multiple

clinical outcomes in CF, frequent assessment of population dynamics is required. However, regular collection and subsequent immediate delivery of fresh sputum samples creates a burden on patients and limits enrollment in such studies. We believe serial home collection and storage of frozen sputum followed en masse by thawing and recovery of isolates offers the potential to overcome this limitation. However, freezing is known to result in a relative reduction in P. aeruginosa populations [13]. If this killing is indiscriminate and no particular phenotype is selected against, then home collection may pose a viable option for the frequent assessment of P. aeruginosa community dynamics. Herein, we sought to determine how freezing affects P. aeruginosa population diversity. Sputum was collected into sterile specimen containers during a routine clinic visit from 10 CF patients with chronic P. aeruginosa infection as defined by the Leeds Criteria [14] (Table 1). No patient was experiencing an exacerbation. Within 3 h of collection, sputum samples were physically sheared using a syringe and an 18-gauge needle, and fractionated. Half of the sputum was processed immediately and serially diluted in Brain Heart Infusion (BHI) broth and plated onto MacConkey (MAC) agar at 10−3 and 10−5 dilutions. The other half, was transferred into a 15 ml sterile conical tube, and placed immediately between two −23 °C rated gel packs (U-tek®, Tegrant Corporation, DeKalb, IL) in a 16 × 16 cm plastic storage container (to accelerate freezing via conduction) and stored in a home freezer for 7 days. Samples were subsequently thawed over 20 min by conduction and serially diluted in BHI in the same fashion. Plates were incubated at 37 °C for 48 h. Colony counts were performed accounting for dilution to calculate a mean CFU/ml per sputum. Eighty-eight isolates were then randomly selected from each sputum sample and stock of each isolate stored in separate wells of 96-well plates. Isolates derived from fresh sputum were termed the immediate community (IC) while those derived from frozen sputum were the frozen community (FC). Four strains were used as controls in each 96-well plate: two lab strains: PA01, ATCC 27853 and two clinical strains: one isolate of the Prarie Epidemic Strain (PES) P47 and one of the Liverpool Epidemic Strain (LES) 299. Similarly, four

Table 1 Patient characteristics from whom samples were derived. Patient ID

1

2

3

4

5

6

7

8

9

10

Gender Mutn_1 Mutn_2 FEV1 (L) FVC (L) BMI (kg/m2) Antibiotics in prior month Colony types_IC Colony types_FC

F F508del F508del 1.2 2.4 16.2 CRO, ATM (I), TOB (I), DOX LG_PK, MUC

M F508del F508del 1.8 3.74 24.9 AZM, AMX, ATM SM_PK, LG_PK, MUC SM_PK, LG_PK, MUC

M F508del F508del 2.14 3.28 26.04 TOB (I)

F F508del F508del 2.79 3.9 20.7 AZM, CRO, ATM (I) PK_MUC, SM

F F508del F508del 0.93 1.84 19.1 TOB (I), ATM (I), AZM SM_PK

M F508del F508del 2.05 3.71 17.5 AZM, TOB (I) PK_MUC, CLR PK_MUC, CLR

F F508del F508del 1.75 3.74 19 AZM, TOBI (I), ATM (I) SM_PK, SM

F F508del P67L 2.02 2.93 25.4 AZM, TOB (I) SM_PK, SM SM_PK, SM

M F508del F508del 3.55 6.24 21 AZM

F M1101K M1101K 2.31 3.01 19.7 AZM, ATM

SM_PK, SM SM_PK, SM

LG_PK, MUC LG_PK, MUC

LG_PK, MUC

PK, MUC, DK_PK PK, MUC, DK_PK

PK_MUC, SM SM_PK

SM_PK, SM

M = males, F = female, IC = immediate community, FC = frozen community. Antibiotic abbrevations: AMX = amoxicillin, CRO = ceftriaxone, ATM = aztreonam, DOX = doxycycline, AZM = azithromycin, TOB = tobramycin, I = Inhaled. Colony morphology abbreviations: LG = large, SM = small, PK = pink, CLR = clear, DK = dark, MUC = mucoid, _ = when a colony type is indicated by two descriptors. Please cite this article as: Poonja A, et al, Effect of freezing sputum on Pseudomonas aeruginosa population heterogeneity, J Cyst Fibros (2017), http://dx.doi.org/ 10.1016/j.jcf.2017.01.004

3

Fig. 1. Box plots comparing (A). intra-patient and (B). inter-patient (cumulative) distributions of virulence factor activity. Virulence factors selected assessed include siderophore, protease, hemolysis and rhamnolipid.

A. Poonja et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx

Please cite this article as: Poonja A, et al, Effect of freezing sputum on Pseudomonas aeruginosa population heterogeneity, J Cyst Fibros (2017), http://dx.doi.org/ 10.1016/j.jcf.2017.01.004

4

A. Poonja et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx

Fig. 2. Distribution of randomly identified P. aeruginosa isolates as a function of MIC range against: A). Aztreonam (ATM); B). Ciprofloxacin (CIP); C). Tobramycin (TOB); D). Meropenem (MEM). Please cite this article as: Poonja A, et al, Effect of freezing sputum on Pseudomonas aeruginosa population heterogeneity, J Cyst Fibros (2017), http://dx.doi.org/ 10.1016/j.jcf.2017.01.004

A. Poonja et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx

testing through a Bonferroni correction. Statistical analysis was carried out using STATA 13.1 (College Stn., Texas). Freezing resulted in a median reduction in P. aeruginosa density of 103 CFU/ml, without eliminating specific morphotypes (Table 1). In similar experiments from five patients where sputum samples were split without homogenization, freeze killing was less profound (mean reduction of 101 CFU/ml) and not improved by mixing whole sputum with equal parts 20% skim milk or 30% glycerol (data not shown). All patients had statistical differences in at least one (of four) virulence factor assessed between IC and FC, with at least one (of four) virulence factor being no different (Fig. 1A). However, no consistent pattern was observed. When virulence factors between IC and FC amongst the entire cohort were compared, no significant differences were noted between the mean diameter of FC relative to the mean diameter of IC for PRO (0.12 mm [95% CI − 0.15 mm to 0.38 mm], p = 0.38), CBA (− 0.17 mm [95% CI −0.41 mm to 0.09 mm], p = 0.19) and RHA (− 0.09 mm [95% CI − 0.26 mm to 0.09 mm], p = 0.30), but modest differences for CAS were present (0.84 mm [95% CI 0.61 mm to 1.06 mm], p b 0.001) (Fig. 1B). None of the patients were observed to have significant differences in proportions of antibiotic resistant isolates between IC and FC (Fig. 2A–D). Inter-patient analysis comparing mean proportions of resistant isolates amongst the entire cohort showed no significant difference between FC and IC: ATM (p = 0.99), CIP (p = 0.20), MEM (p = 0.26), and TOB (p = 0.46). All results were assessed with a conservative adjustment for multiple testing done at an α of 0.05 reflecting a p b 0.006 using a Bonferroni correction. Here we confirmed that P. aeruginosa populations chronically infecting CF lungs are highly heterogeneous and that intra-sample phenotypic diversity is underestimated if only few colonies are analyzed per sample [5–9]. Freezing of samples resulted in a modest yet non-selective reduction of P. aeruginosa population density. However, while intra sample diversity was noted in IC and FC, no individual phenotype when assessed in aggregate was selected against by freezing. Our results are limited by the fact that sputum itself is tremendously heterogeneous. Our sputum sample was separated into two equal portions by physical shearing, one half being processed immediately (IC), and the other half being processed post 1-week freezing (FC). This process may have been imperfect at disrupting bacterial aggregates, and thus collections of particular phenotypes. In fact, studies assessing concordance between different methods of respiratory sample collection (i.e. sputum/throat swabs and bronchoalveeolar lavage) have in the past always attributed differences to the means of sample collection [18,19]. Indeed, it may even be that heterogeneity exists between simultaneously collected samples assessed using the same methodology – warranting replicate samples in such studies. The dynamics of P. aeruginosa populations and its impact on clinical events (i.e. pulmonary exacerbations), outcomes, and treatment response is hereto unknown. Our study suggests home collection may pose a viable means by which to frequently monitor

5

P. aeruginosa population dynamics in CF lung disease to answer some of these questions. References [1] Razvi S, Quittell L, Sewall A, Quinton H, Marshall B, Saiman L. Respiratory microbiology of patients with cystic fibrosis in the United States, 1995–2005. Chest 2009;136:1554–60. [2] Lambiase A, Raia V, Del Pezzo M, Sepe A, Carnovale V, Rossano F. Microbiology of airway disease in a cohort of patients with cystic fibrosis. BMC Infect Dis 2006;6:4. [3] Kosorok MR, Zeng L, West SE, Rock MJ, Splaingard ML, Laxova A, et al. Acceleration of lung disease in children with cystic fibrosis after Pseudomonas aeruginosa acquisition. Pediatr Pulmonol 2001;32(4):277–87. [4] Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL. Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 2002;34(2):91–100. [5] Foweraker JE, Laughton CR, Brown DF, Bilton D. Phenotypic variability of Pseudomonas aeruginosa in sputa from patients with acute infective exacerbation of cystic fibrosis and its impact on the validity of antimicrobial susceptibility testing. J Antimicrob Chemother 2005;55(6):921–7. [6] Workentine ML, Sibley CD, Glezerson B, Purighalla S, Norgaard-Gron JC, Parkins MD, et al. Phenotypic heterogeneity of Pseudomonas aeruginosa populations in a cystic fibrosis patient. PLoS One 2013;8(4):e60225. [7] Folkesson A, Jelsbak L, Yang L, Johansen HK, Ciofu O, Hoiby N, et al. Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nat Rev Microbiol 2012;10(12):841–51. [8] Clark ST, Diaz Caballero J, Cheang M, Coburn B, Wang PW, Donaldson SL, et al. Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis. Sci Rep 2015;5:10932. [9] Winstanley C, O'Brien S, Brockhurst MA. Pseudomonas aeruginosa evolutionary adaptation and diversification in cystic fibrosis chronic lung infections. Trends Microbiol 2016;24(5):327–37. [10] Smith AL, Fiel SB, Mayer-Hamblett N, Ramsey B, Burns JL. Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis. Chest 2003; 123(5):1495–502. [11] Parkins MD, Rendall JC, Elborn JS. Incidence and risk factors for pulmonary exacerbation treatment failures in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa. Chest 2012;141(2):485–93. [12] Lam JC, Somayaji R, Surette MG, Rabin HR, Parkins MD. Reduction in Pseudomonas aeruginosa sputum density during a cystic fibrosis pulmonary exacerbation does not predict clinical response. BMC Infect Dis 2015;15:145. [13] Williams DL, Calcott PH. Role of DNA repair genes and an R plasmid in conferring cryoresistance on Pseudomonas aeruginosa. J Gen Microbiol 1982;128(1):215–8. [14] Lee TW, Brownlee KG, Conway SP, Denton M, Littlewood JM. Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. J Cyst Fibros 2003;2(1):29–34. [15] Pinzon NM, Ju LK. Analysis of rhamnolipid biosurfactants by methylene blue complexation. Appl Microbiol Biotechnol 2009;82(5):975–81. [16] Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9(7):676–82. [17] Performance standards for antimicrobial susceptibility testing. In: (CLSI) CaLSI, editor. Nineteenth Informational Supplement; 2009. p. 1–156. [18] Seidler D, Griffin M, Nymon A, Koeppen K, Ashare A. Throat swabs and sputum culture as predictors of P. aeruginosa or S. aureus lung colonization in adult cystic fibrosis patients. PLoS One 2016;11(10):e0164232. [19] Baughman RP, Keeton DA, Perez C, Wilmott RW. Use of bronchoalveolar lavage semiquantitative cultures in cystic fibrosis. Am J Respir Crit Care Med 1997;156(1):286–91.

Please cite this article as: Poonja A, et al, Effect of freezing sputum on Pseudomonas aeruginosa population heterogeneity, J Cyst Fibros (2017), http://dx.doi.org/ 10.1016/j.jcf.2017.01.004