Applied Radiation and Isotopes 77 (2013) 84–88

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Applied Radiation and Isotopes

0969-80

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Characterization of non-tuberculosis mycobacteriaby neutron radiography

Jaqueline M. da Silva a, Verginia Reis Crispim a,n, Marlei Gomes da Silva b,Vanessa Rodrigues Furtado c, Rafael Da Silva Duarte b

a Programa de Engenharia Nuclear/COPPE/CT/UFRJ, Av. Horacio Macedo, 2030, Bloco G, sala 206, Cidade Universitaria, 21.941-914 Rio de Janeiro, RJ, Brasilb Instituto de Microbiologia Professor Paulo de Goes/CCS/UFRJ, Av. Carlos Chagas Filho, 373. CCS-Bloco I, Cidade Universitaria, 21.941-902 Rio de Janeiro,

RJ, Brasilc Instituto de Quımica/CT/UFRJ, Av. Athos da Silveira Ramos, 149 Bloco A-51 andar, Cidade Universitaria, 21941-909 Rio de Janeiro, RJ, Brasil

H I G H L I G H T S

c We aimed to characterize morphologically nontuberculous mycobacterium by neutron radiography.c This methodology greatly reduced the time to identify of NTM compared to conventional microbiological assays.cImages of NMT resulted magnified 1000x and are displayed on a conventional optical microscope.cDoping with PAMAM dendrimer and sodium borate improved the visualization of NTM by NR.

a r t i c l e i n f o

Article history:

Received 28 November 2012

Received in revised form

6 February 2013

Accepted 13 February 2013Available online 26 February 2013

Keywords:

Non-tuberculosis mycobacteria

M. fortuitum

Neutron radiography

CR-39

Dendrimers

43/$ - see front matter Published by Elsevier

x.doi.org/10.1016/j.apradiso.2013.02.009

esponding author. Tel.: þ55 2121 25628439

ail addresses: vrcrispim@gmail.com (V.R. Cri

@iq.ufrj.br (V.R. Furtado), rafaelsilvaduarte@g

a b s t r a c t

The genus Mycobacterium shares many characteristics with Corynebacterium and Actinomyces genera,

among which the genomic guanine plus cytosine content and the production of long branched-chain fatty

acids, known as mycolic acids are enhanced. Growth rate and optimal temperature of mycobacteria are

variable. The genus comprises more than 140 known species; however Mycobacterium fortuitum, a fast

growing nontuberculous mycobacterium, is clinically significant, because it has been associated to several

lesions following surgery procedures such as liposuction, silicone breast and pacemaker implants, exposure

to prosthetic materials besides sporadic lesions in the skin, soft tissues and rarely lungs. The objective of the

present study is to reduce the time necessary for M. fortuitum characterization based on its morphology and

the use of the neutron radiography technique substituting the classical biochemical assays. We also aim to

confirm the utility of dendrimers as boron carriers. The samples were sterilized through conventional

protocols using 10% formaldehyde. In the incubation process, two solutions with different molar ratios

(10:1 and 20:1) of sodium borate and PAMAM G4 dendrimer and also pure sodium borate were used. After

doping and sterilization procedures, the samples were deposited on CR-39 sheets, irradiated with a

4.6�105 n/cm2 s thermal neutron flux for 30 min, from the J-9 irradiation channel of the Argonauta IEN/

CNEN reactor. The images registered in the CR-39 were visualized in a Nikon E400 optical transmission

microscope and captured by a Nikon Coolpix 995 digital camera. Developing the nuclear tracks registered

in the CR-39 allowed a 1000� enlargement of mycobacterium images, facilitating their characterization,

the use of more sophisticated equipment not being necessary. The use of neutron radiography technique

reduced the time necessary for characterization. Doping with PAMAM dendrimer improved the visualiza-

tion of NTM in neutron radiography images.

Published by Elsevier Ltd.

1. Introduction

Mycobacteria belong to Mycobacterium, sole genus of theMicobacteriaceae family (Brenner et al., 2005). These organisms

Ltd.

; fax: þ55 2121 25628444.

spim),

mail.com (R.S. Duarte).

are pleomorphic, aerobic or microaerophile, motionless, do nothave capsule or produce spores, straight or slightly curved, rod-shaped with size ranging from 0.2 to 0.6 mm width and from1.0 to 10 mm length (Holt et al., 1994).

The cell wall of mycobacteria is composed of four layers. The innerlayer is a peptidoglycan composed by N-glycolilmuramic acid. Thefollowing layer is composed of arabinogalactan. Mycolic acids formthe third layer and are responsible for 60% of the cell wall. The outerlayer is formed by different lipids, including glycolipids, sulfolipids,

J.M. da Silva et al. / Applied Radiation and Isotopes 77 (2013) 84–88 85

phenolic glycolipids, peptide-glycolipids and lipid-arabinomanan(Rastogi, 1991).

In relation to multiplication time they are classified as fast or slowgrowing bacteria. The temperature for bacterial growth ranges from25 1C to 45 1C. Colonies can have rough or smooth aspect and somepresent carotenoid pigments, usually of orange or yellow color in thepresence or absence of light (Wayne and Kubica, 1986).

Currently, Mycobacterium genus encompasses 142 species and 11sub-species, described in the list of bacterial species with approvednames (Euzeby, 1998). Some of those species were classified accord-ing their pathogenicity: strictly pathogenic, pathogenic, potentiallypathogenic, and rarely pathogenic and saprofites (Tsukamura, 1984).

Non-tuberculosis mycobacteria (NTM), classified as potentiallypathogenic or rare pathogens include several species that maycause pulmonary, ganglionary and cutaneous infections anddisseminated infections in immune compromised individuals.(NTM), classified as potentially pathogenic or rare pathogensinclude several species that may cause pulmonary, ganglionaryand cutaneous infections and disseminated infections in immunecompromised individuals.

A methodology based on nuclear assays and specific tracerswas adopted to characterize M. fortuitum in positive samples. Thepresent study aims at speeding up the process, in relation to thetime spent by conventional biochemical methods, and conse-quently reduce the time between diagnosis and beginning oftreatment.

M. fortuitum is considered cosmopolite, widely found in waterand soil samples collected around the world. As environmentalbacterium, it is less virulent than M. tuberculosis, but when itestablishes a pathogenic process, during an infection, it is difficultto eradicate because it presents a great resistance to antimicro-bials (Palwade et al., 2006).

Among the non-destructive test techniques (NDT), neutronradiography appears as a powerful tool in imaging. Neutronradiography requires an appropriate neutron beam, an object tobe neutron radiographed and a detection device. The secondaryradiation, generated by nuclear reaction between neutrons, theflux of which is modulated by the absorbent and scatteringcharacteristics of the object and the converter nucleus, ionizesthe plastic detector and forms nuclear tracks. When the converteris boron, the greater the amount of boron incorporated to themycobacteria, the greater the number of nuclear tracks of alphaparticles generated in the nuclear reaction 10B(n, a)7Li, in theregion occupied by each mycobacterium, in comparison to thatfound in the plastic film. In this manner, the visualization of themycobacteria contained in the sample, becomes very clear in theneutron radiography image.

The presence of hydrogenated material in the composition ofmycobacteria may reduce the probability of a particles ionizing theCR-39. Thus, in order to obtain clear neutron radiography images it isnecessary to improve the incorporation of the converter agent.Therefore, a solution of PAMAM G4-type dendrimers was used withsodium borate, the chosen converter agent. Among various applica-tions of dendrimers, drug delivery in their core cavities or outer layerhas been given great attention in medicine (Beezer et al., 2003).Dendrimers are three-dimensional, polymeric macromolecules thatmay me hyper branched and composed of a core. Dendrimers can bechemically synthetized by the divergent or the convergent methods(Bernhardsson and Shishoo, 2003). In the divergent method, thebranched units are added up one by one, from the core to the end,thus multiplying the number of periphery groups in their dendriticstructure. The convergent method follows the opposite way, theskeleton is constructed starting in the periphery and elaborating tothe core (Bosman et al., 1999). Currently, two types of dendrimers arecommercially available: poly(amine)amine (PAMAM) and Poly-(propylene)imines (PPI).

2. Materials and methods

The experimental procedure adopted in the neutron radio-graphy technique comprised five stages: pre-attack, sample pre-paration, irradiation, chemical attack, and image visualization.

The pre-attack consisted in slightly corroding the surface ofCR-39 sheets, to increase the adherence of mycobacteria.The sheets were placed in test tubes containing 6.25 N NaOHand kept in a thermo regulated bath unit, at 90 1C for 15 min.Next, the sheets were washed with tap water and distilled waterand then dried with the help of a hairdryer.

For sample preparation 0.1 ml of M. fortuitum sample, pre-cultured at 37 1C for 7 days in 7H9 culture media, was used. Afterculture, the samples were suspended in enough distilled water withsterile glass beads to obtain a turbidity equivalent to tube 1 of theMac Farland scale; and then this suspension was diluted to3.0�105 CFU/ml.

For sample inactivation, 50 ml of the bacterial solution wereplaced in a microtube containing the same amount of 10%formaldehyde. After automatic stirring for 30 s, the sample wasleft to stand at room temperature for 60 min. After this period, thedoping with the converter was carried out, adding 10 ml of sodiumborate, in the first procedure, and 10 ml of the sodium borate/PAMAM G4 dendrimer solution in molar ratios 10:1 and 20:1. Theincubation temperatures used were 4 1C and 37 1C and theincubation times 30, 60 and 120 min. After the doping processwas concluded, a 5.0 ml sample was placed in each CR-39 sheetand dried at 50 1C for 10 min.

During irradiation, the detectors were fixed on an aluminumplate along the edge of the J-9 channel of the Argonauta/IEN/CNEN reactor for 30 min. The thermal neutron flux and meanenergy were 4.46�105 n cm�2 s�1 and 30 meV, respectively.

The reaction 10B(n,p)7Li results in alpha (a) particle tracks withdiameter on the order of Angstroms in the solid-state detectors. Inorder to be visualized in an optical microscope, the latent tracksregistered in the CR-39 sheets were submitted to a chemicalattack in a thermo regulated bath. The detectors were placed intest tubes containing 6.25 N NaOH (6.25 N) for 60 min at 90 1C.The washing and drying processes were similar to the pre-attack.

Finally, the neutron radiography images registered on the CR-39 sheets were visualized in a Nikon E400 optical transmissionmicroscope coupled to Nikon Coolpix 995 digital camera, whichcaptured the digital images.

3. Results and discussion

M. fortuitum culture was carried out at 37 1C, during seven days, in7H9 culture media. After growing, the microorganisms were left tostand for 60 min, in 10% formaldehyde, at room temperature forinactivation. Doping was performed with pure sodium borate andwith 10:1 and 20:1 (molar ratio) sodium borate/PAMAM G4 den-drimer solutions. Irradiation was conducted after 5 ml sample weredeposited on CR-39 sheets. Then, they were chemically attacked with6.25 N NaOH at 90 1C, for 60 min. The images were obtained aftersheet washing and drying.

In the first experiment, only sodium borate was used forbacillus doping. It was expected to observe around 1000 myco-bacteria in each CR-39 sheet. In order to assess the influence ofincubation temperature and time, the incubation was performedat 4 1C and 37 1C and for 30, 60 and 120 min.

Fig. 1 shows M. fortuitum neutron radiography images afterdoping with sodium borate at 4 1C, for 30, 60 and 120 min.

The images in Fig. 1 present structures morphologically similarto bacilli, but the amount was lower than expected, probablybecause the incubation temperature did not favor microorganism

Fig. 1. Images of M. fortuitum deposited on CR-39, doped with 10 ml sodium borate and incubated at 4 1C, for variable time intervals: (a) 30 min, (b) 60 min and

(c) 120 min. The arrows indicate isolated bacilli and the circles CR-39 manufacture defects.

Fig. 2. Images of M. fortuitum deposited on CR-39, doped com 10 ml of sodium borate and incubated at 37 1C, for different incubation time: (a) 30 min, (b) 60 min and

(c) 120 min. The arrows show isolated or clumped bacilli.

Fig. 3. Morphologic characterization of M. fortuitum, when doped at 4 1C, with 10:1 (molar ratio) sodium borate/PAMAM G4 dendrimer solution. The incubation times

were: (a) 30 min, (b) 60 min and (c) 120 min.

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doping with boron. It is important to highlight that the interac-tion between the neutrons and boron is essential for the occur-rence of the nuclear reaction and consequently for obtainingneutron radiography images.

The neutron radiography images after doping with sodiumborate at 37 1C, at the same time intervals of 30, 60 and 120 min,are shown in Fig. 2.

The neutron radiography images of M. fortuitum doped, at37 1C, with sodium borate, for the three time intervals, 30, 60 and

120 min, show a greater number of bacilli than shown in Fig. 1,when the microorganisms were doped at 4 1C, and the remainingparameters maintained. Fig. 2 shows isolated and clumped bacilli,while Fig. 1 shows just one or two isolated bacilli.

Aiming at separating the clumped bacilli, shown in theneutron radiography images, which means count increasing aswell as improving the sharpness of the images, a second experi-ment was conducted using solutions of sodium borate andPAMAM dendrimer, in molar ratios 10:1 and 20:1. Fig. 3 shows

Fig. 4. Morphologic characterization of M. fortuitum, when doped with 10:1 and 20:1 (molar ratio) sodium borate/PAMAM G4 dendrimer (a) M. fortuitum agglomerates

after doping with 10:1 molar ratio solution at 37 1C for 60 min, and (b) isolated bacilli after doping with 20:1 Molar ratio solution at 4 1C for 30 min.

Fig. 5. Morphologic characterization of M. fortuitum, when doped at 37 1C, with 20:1 (molar ratio) sodium borate/PAMAM G4 dendrimer solution. The images, (a), (b) and

(c), present isolated or clumped bacilli after incubation for 30, 60 and 120 min, respectively.

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images of M. fortuitum doped at 4 1C, with 10:1 sodium borate andPAMAM dendrimer solution for incubation times of 30, 60 and120 min, respectively.

The neutron radiography images obtained with the use ofdendrimer show the presence of tens of bacilli scattered on theCR-39 sheets and clumped microorganisms as shown in Fig. 2 arenot observed. Fig. 3(b) has not the same enlargement of figures(a) and (c), because, since it presents a much higher number ofbacilli, the real count of scattered mycobacteria would beimpaired.

Similar images were not obtained when the concentrations10:1 and 20:1 were employed at 37 1C and 4 1C respectively, i.e., itwas not possible to visualize the expected amount of isolatedbacilli, as shown in Fig. 4.

The images of the last procedure, when 20:1 (molar concentra-tion) sodium borate/PAMAM solution was used to dope M. fortuitum,at 37 1C, for the same previous time intervals present clumped orisolated bacilli, besides roughness in CR-39 sheet. Fig. 5 presents theimages obtained for the three incubation times: 30, 60 and 120 min.

The images in Fig. 5 also show damages and roughness inCR-39 sheet. This may have been caused by an unexpecteddevelopment temperature increase which occurred because of atemporary failure in the thermo regulated bath unit.

It is important to highlight the excellent quality of the images,which represent two-dimensional projections of isolated orclumped bacilli visualized in a conventional transmission opticalmicroscope.

4. Conclusions

By doping the samples of M. fortuitum with sodium borate, atthe temperature of 37 1C, allowed the visualization of highernumber of isolated or clumped bacilli than doping at 4 1C.

Most of the neutron radiography images of M. fortuitum dopedwith sodium borate and PAMAM G4 dendrimer presented ahigher number of isolated or clumped bacilli than in the imagesobtained without using the dendrimer, thus confirming theefficiency of its use as boron carrier.

The temperature and incubation time that resulted in animage with higher number of isolated bacilli were 4 1C and60 min, respectively, and the molar ratio of the sodium borate//PAMAM solution was 10:1. However the image with the highernumber of clumped bacilli was obtained when M. fortuitum wasdoped at 37 1C, for 30 min with solution at 20:1 molar ratio.

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The results show that the technique provides images enlarged1000� and thus facilitates mycobacterium identification without theneed for sophisticated equipment such as a scanning electronmicroscope. The technique also allowed reducing the time for bacillusidentification, when compared to the time demanded by othermethodologies.

Acknowledgments

To the Laboratory of Mycobacteria/CCS/UFRJ for providingM fortuitum samples and technical support.

To Prof. Vanessa, Head of the IQ/UFRJ Chemistry Laboratory forsupplying PAMAM G4 and information on dendrimer use.

To the Reactor Department/DIREA/IEN/CNEN for making avail-able the irradiation of microbiological samples.

To the CNPq for financial support for research.

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