Title: The impact of relative humidity and collection media on mycobacteriophage D29 aerosol ...

33 Pages·2011·1.46 MB·English

AEM Accepts, published online ahead of print on 22 December 2011
Appl. Environ. Microbiol. doi:10.1128/AEM.06610-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
1 Title:
2 The impact of relative humidity and collection media on mycobacteriophage D29 aerosol
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4 Authors/Affiliations:
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5 Keyang LIU, Zhanbo WEN, Na LI, Wenhui YANG, Jie WANG, Lingfei HU, Xiaokai DONG, Jianchun LU, w
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6 Jinsong LI* a
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8 State Research Center for Bio protective Equipment & Engineering Technology, State Key Laboratory of
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9 Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s :/
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10 Republic of China
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12 Address: /

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13 Department of Biosafety, Beijing Institute of Microbiology and Epidemiology, 20# Dongda Street, 100071 A
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14 Fengtai District, Beijing, People’s Republic of China 1
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16 Contact: by

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17 Corresponding author: e
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18 Jinsong LI
19 Email: [email protected]
20 Tel: [phone]
21 Fax: [phone]
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22 First author:
23 Keyang LIU
24 Email: [email protected]
25 Tel: [phone]
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27 Running title: bacteriophage D29 generation and sampling a
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29 Word count:
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30 Abstract: 254 :/
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31 Text: 4299
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33 Abstract
34 This study was conducted to evaluate the effect of aerosol generation, methods of sampling, storage
35 conditions and relative humidity on the culturability of the mycobacteriophage D29. The lytic phage D29
36 can kill Mycobacterium tuberculosis and the phage aerosol can be treated as a potential tool for
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37 tuberculosis treatment. The culturability of D29 was tested using a test chamber designed for the w
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38 bioaerosols research against three spray liquids (deionized water, PBS and normal saline), four collection a
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39 media (suspension medium (SM), nutrient broth, PBS and deionized water), two sampling systems (the
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40 all-glass impinger AGI-30 and the Biosampler) and across a range of humidities (20% - 90%). The effect
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41 of storage conditions on the culturability of collected sample was also evaluated for the AGI-30 impinger. :/
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42 The results proved that viable phage D29 particles generated by deionized water were approximately
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43 30-fold and 300-fold higher than PBS and normal saline, respectively. As collection media, SM buffer and
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44 nutrient broth were observed to yield a higher number of plaques compared to PBS and deionized water. /

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45 No difference was observed in collection efficiency between AGI-30 and Biosampler with two detection A
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46 methods (culture-based technique and real-time PCR). The culturability of collected D29 in SM buffer or 1
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47 nutrient broth can be maintained up to 12h irrespective of storage temperature. Relative humidity was 0
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48 found to strongly influence airborne D29 culturability which is 2-20 folds higher in low humidity (25%) by

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49 than middle (55%) or high humidity (85%). This research will help identify the optimal means for the e
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50 application of D29 aerosol in animal inhalation experiments.
51 Keywords: mycobacteriophage, bioaerosol, spray liquid, sampling media, Biosampler, real-time PCR
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53 1. Introduction
54 Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, was responsible for over 9
55 million new cases and almost 2 million deaths in 2008 alone (66). Approximately 15% of the total
56 population in Afghanistan is infected with tubercle bacillus (13). Together with the emergence of
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57 multi-drug resistant and extensively-drug resistant strains, and the human immunodeficiency virus (HIV) w
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58 epidemic, tuberculosis is becoming one of the most significant threats to global health (67). a
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59 The use of bacteriophage as an alternative to antibiotic therapy for bacterial disease is being reappraised
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60 in the Western world because of the emergence of multi-drug resistant bacterial infections (7, 54). Phage
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61 therapy dates back to 1919, when d’Herelle (12) first treated an outbreak of avian typhosis among farmed :/
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62 chickens. However, the approach was abandoned in the west following the advent of clinical antibiotic
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63 treatment (25, 58). To date, application of phage technologies in the field of treatment of various bacterial
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64 infections has shown significant success in studies in Eastern Europe and is still used today to treat /

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65 patients (19, 29, 30, 56). Recently, studies in animal models have demonstrated the effects of A
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66 bacteriophage therapy on lung infections (6, 10). Although a large number of M. tuberculosis 1
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67 bacteriophages have been oberserved and complete genomes are now available (22), few published 0
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68 studies have described the use of them in therapy experiments (4, 23, 33, 57, 69). Work with by

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69 mycobacteriophage mostly concentrates on DS-6A, D29 and TM4 (35). The high specificity of DS-6A e
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70 makes it an attractive candidate for anti-tuberculosis therapy while D29 and TM4 have been used in tests
71 for diagnosis and drug susceptibility (35, 38). Early phage therapy experiments not only failed to cure
72 experimentally infected guinea pigs, but had an adverse effect on their survival (23, 33). In 1991
73 Zemskova and Dorozhkova reported that DS-6A could have a beneficial effect in guinea pigs with
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74 disseminated tuberculosis, but its action was observed to be less than that of isoniazid monotherapy (70).
75 In 2002 Broxmeyer et al. proved that the lytic phage TM4 can kill intracellular pathogens (M.avium and
76 M.tuberculosis) when delivered by a nonvirulent mycobacterium (4). However, no further report about the
77 therapeutic effect of TM4 can be found. In 2006, Peng et al. reported that phage D29 can kill the
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78 intracellular M.tuberculosis in vitro without any delivery system (47). D29, a lytic phage, is one of the w
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79 most commonly used mycobacteriophage for tuberculosis diagnosis (8, 9, 11, 16, 17, 36, 37, 39, 55, 69). a
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80 It is able to infect both slow-growing pathogenic mycobacteria such as M.tuberculosis and fast-growing
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81 environmental strains, and will form visible plaques following overnight incubation on a lawn of
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82 fast-growing M.smegmatis (38). Its potential as a therapeutic tool is worthy of being investigated. :/
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83 The potential of phages as antibacterial agents lies in their ability to kill host cells at the end of an
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84 infectious cycle (45). However, phage therapy may fail if insufficient number of phage particles reaches
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85 the target organs to trigger the infection cycle (45, 46). Since tuberculosis is primarily a disease of the /

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86 lung (43), its treatment by phage aerosols can be considered. Recently phage aerosol has been used to A
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87 treat respiratory E. coli infections in experimentally contaminated birds (46). Clinical trials are also 1
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88 considered for an aerosol variant for use in patients with cystic fibrosis (www.biocontrol-ltd.com). 0
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89 Unfortunately, on the one hand, there is no evidence that aerosol delivery of phage would result in more by

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90 phage reaching the lung than other routes such as injection and intranasal. On the other hand, phages tend e
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91 to be rapidly inactivated by the reticuloendothelial system but it is unknown whether phages delivered by
92 aerosol would be retained longer in the lung (41, 64). Well designed animal exposure systems are needed
93 to address these questions.
94 In order to better conduct animal inhalation studies, the properties of D29 aerosol have to be understood.
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95 Phage D29 with its long tail is fragile and the culturability of its aerosol may be affected by many factors,
96 such as the relative humidity (RH), temperature, aerosolization medium and sampling methods.
97 Accordingly, in order to better understand the characteristics of the D29 aerosols and choose optimal
98 conditions for survival in aerosols, we examined methodological influences of generation, sampling,
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99 storage conditions and RH on D29 culturability. w
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100 a
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101 2. MATERIAL AND METHODS f
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102 2.1 phage suspension
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103 Bacteriophage D29 was kindly provided by Guozhi WANG, National Institute for the Control of :/
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104 Pharmaceutical and Biological Products, China.
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105 The bacterial host M.smegmatis mc2155 cells were propagated in 7H9 Broth (DifcoTM Middlebrook m
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106 7H9 Broth, BD; MI) for 60 h. Approximately 104 plaque forming units (PFU) D29 phages were mixed /

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107 with 300 µl bacteria stock and 3-5 ml 7H9 top agar (MI + 0.7% agar). The mixture was plated on a 90 mm A
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108 Petri dish containing Middlebrook 7H10 agar (DifcoTM Middlebrook 7H10 Broth, BD). Ten such plates il 1
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109 were prepared and incubated overnight at 37 ºC. Both 7H9 and 7H10 broth contained 10% (vol/vol) 0
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110 OADC (BBLTM Middlebrook OADC Enrichment, BD) which was added when temperature of the by

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111 medium was below 60 ºC. After cultivation, the plates showed confluent lysis. Top agar in these plates e
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112 were scraped to a tube, followed by pouring 10 ml suspension medium (SM, containing 100mM NaCl,
113 8.5mM MgSO4﹒7H2O, 50mM Tris.Cl (PH 7.5) and 0.01% gelatin) into it (50). The tube was centrifugated
114 at 9000 × g for 10 min at 4 ºC to remove bacterial cells (42), and the supernatant containing phage was
115 stored at 4 ºC. The plaque assay described by Adam (1) was used to measure the bacteriophage
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116 concentration.
117 2.2 Aerosol generation and test system
118 The test chamber which has a volume of 2.06 m3 is connected to the generation, sampling, autocontrol
119 equipment and auxiliary ports. The schematic diagram of the system is shown in Figure 1. Pipes
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120 transporting airstreams are connected with the left side of the test chamber. Dry, clean and compressed w
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121 laboratory air is divided into two streams, one is used for nebulization and the other for supplement or a
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122 dilution. The flow rates of both airstreams are monitored and controlled with respective glass rotameters.
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123 Aerosolization of phage D29 was achieved with the Collison 6-jet nebulizer (BGI Inc., Waltham, MA)
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124 into the chamber at a constant flow rate of 10 L min-1 with dry and filtered air. This nebulizer is the gold :/
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125 standard for laboratory bioaerosol generation (49). The concentration of phage D29 suspension in the
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126 nebulizer was 10 PFU/ml.
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127 The AGI-30 sampler, an all glass instrument with a curved inlet that has a 50% cut-size of 0.3µm (44), /

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128 was used to collect aerosols. The impingers were filled with 20 ml sampling media (containing 0.005% A
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129 antifoam and 0.01% Tween80) and operated with a flow pump called MS-Ⅰ. The flow pump was 1
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130 developed by our lab, and the flow rate and sampling time in this experiment were set at 12.5 L min-1 and 0
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131 5 min, respectively. The antifoam used in the experiments was olive oil. by

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132 Once the nebulizer was turned on, the aerosol was allowed to equilibrate inside the chamber for a e
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133 generation time of 5 min before beginning of the 5 min sampling. The total generation time is 10 min. A
134 port was used to supply the chamber with make-up air at a flow rate of 50 L min-1. When one experiment
135 is finished, the remaining aerosol is filtered and expelled to environment.
136 An aerodynamic particle sizer (APS, Model 3321, TSI, Inc.) was used to measure the particle size
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137 distribution of the aerosol. The aerosol generated is described by the MMAD (mass median aerodynamic
138 diameter) and GSD (geometry standard deviation). MMAD is the particle diameter that halves the mass
139 distribution, and GSD provides a representation of the uniformity of the particles (59).
140 The temperature and relative humidity sensors are fixed in the inner wall and monitor the temperature
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141 and relative humidity. When the humidity is below the set value, the humidifier in the test chamber runs w
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142 automatically. The temperature cannot be changed and is equal to the room temperature (25 ± 2 ºC). a
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143 2.3 Test procedures f
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144 The optimal spray liquid was first evaluated based on the fraction of culturable phage particles in the
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145 sampled aerosol to that in the suspension preaerosolization. Because this test was not conclusive, the :/
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146 spray medium that resulted in highest fraction of culturable D29 was selected in the following
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147 experiments. This generation method was used to test for the most efficient collection fluid, sampler, and
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148 the influence of relative humidities on phage D29 culturability. /

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149 To increase the output of aerosol containing viable phage particles in animal exposure experiments, the A
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150 ability of D29 to endure the aerosolization was evaluated. Three solutions containing no organic 1
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151 molecules to get rid of potential injury to animal respiratory tract were chosen as spray liquids. The media 0
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152 were sterile PBS (containing 0.85% NaCl; 0.01mol L-1, PH 7.2), deionized water and normal saline by

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153 (containing 0.85% NaCl). The AGI-30 samplers were used to collect generated aerosols. Each spray e
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154 experiment was performed at least in triplicate.
155 Although there are many sample liquids, four of them were selected to determine the best sampling
156 medium for the D29 aerosols. They were PBS (containing 0.85% NaCl; 0.01mol L-1, PH 7.2), nutrient
157 broth (with 0.5% NaCl), deionized water and SM buffer. Each solution contained 0.01% Tween80 to
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158 provide protection to phage D29 during sampling.
159 The Biosampler (SKC Inc.) is also an all glass instrument, similar in concept and shape to the AGI-30
160 impinger, but with three angled nozzles, causing impingement and centrifugal motions (65). Both the
161 AGI-30 and Biosampler were filled with 20 ml SM buffer and ran simultaneously at the recommended
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162 operational flow rate of 12.5 L min-1 (Ace Glass Inc.). The sampling was lasted for 5 min. After sampling w
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163 completed, each collection liquid containing phage D29 was separated into two equal portions, one of a
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164 which was inoculated for plaque assay and the other treated for real-time PCR. The genome extraction
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165 and real-time PCR assay were both operated using the same protocol as that used to generate a standard
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166 curve. :/
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167 Real-time PCR assays were performed with Line-Gene Real-Time PCR Detection Systems (BIOER
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168 Inc., China). Primers and TaqMan®-MGB probes were designed by Applied Biosystems (ABI Inc.) for the m
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169 detection and amplification of the conserved sequences (GenBank accession number U81553.1). The /

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170 sequences are: forward primer 5’-AGC TAC GCG GGT TCG ATTC-3’, reverse primer 5’-CCG GTC A
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171 CTC GGG CTA AAA-3’ and probe 5’-FAM-CCG TCG CCC GCT CCA-3’-NFQ-MGB. The assay was 1
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172 carried out in 20 µL volumes containing 1 µL 20× TaqMan® Gene Expression Assay (mixture of probe 0
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173 and primes; ABI), 10 µL 2× TaqMan® Gene Expression Master Mix (ABI Inc.), 4 µL DNA template and 5 by

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174 µL RNase-free water. The PCR cycling protocol was as follows: initial step to active polymerase at 95 ºC e
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175 for 10 min, followed by 40 amplification cycles of 95 ºC for 15 s (denaturation) and 60 ºC for 1 min
176 (annealing and extension). Genomic DNA extraction from pure culture of D29 with the TIANamp Virus
177 DNA Kit (TIANGEN Inc., China) was performed according to the manufacturer’s guidelines. Eight serial
178 1:10 dilutions of isolated DNA concentration corresponding to 4.22×107 PFU µL-1 were made, and when
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179 the threshold cycle (CT) for each standard was plotted against the logarithm of starting quantity (PFU) in
180 20-µL reaction volume, the standard curve was obtained.
181 The survivability of the D29 aerosol was evaluated in different RH. After the Collison nebulizer had
182 nebulized for 10min, the generated aerosol in the test chamber was allowed to decay for 15 and 30 min in
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183 low RH (25±5%), middle RH (55±5%) and high RH (85±5%), respectively. Then, AGI-30 samplers filled w
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184 with deionized water were operated to sample the aerosol for 5 min. The effect of RH on the variation in a
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185 phage culturability was determined by calculating the logarithm of recovery rates.
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186 2.4 Calculation of collection efficiency
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187 The total recovery rate (TR) was used to evaluate the culturability and viability of aerosolized D29. TR :/
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188 is defined as Ctest / Csusp, where Ctest is PFU m-3, i.e., the number of infective phage particles in test m.
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189 chamber, and Csusp is PFU ml-1, i.e., the number of infective phage particles present in suspension m.
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190 preaerosolization. Ctest was obtained by adjusting results from the cultured impinger fluids in PFU ml-1 of /
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191 liquid plated for volume of air passed through the samplers and final volume of liquid in the vessel. Then A
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192 TR was calculated as 1
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193 TR= Q ×satm×plCer 019
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194 Where C is plaque forming units in the sampler, Vsampler is final liquid volume in the impinger, Qsampler is gu
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195 sample flow rate, and t is sampling time. t
196 This is technically a unitless parameter, since both Ctest and Csusp are measures of numbers of the phage.
197 Plaque assays were used to define concentration of the phage in the sample media. Each collected
198 sample was serially diluted 10-fold and each solution was plated on duplicate plates and incubated at 37ºC.
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